Novel heteromultimeric ion channel receptor and uses thereof

ABSTRACT

The present invention describes a heteromultimeric proton-gated ion channel (herein called ASIC-2S.2) with distinctive properties. Compositions and methods are provided for producing and expressing functional ASIC-2S.2 channels, composed of ASIC2A and ASIC3 subunits. The invention also provides genetically engineered expression vectors comprising the nucleic acid sequences encoding both ASIC2A and ASIC3 and host cells coexpressing both ASIC2A and ASIC3 subunits. Also provided herein are genetically engineered nucleic acids encoding chimeric proton-gated ion channels comprised of at least two different subunits, as well as expression vectors and host cells comprising said engineered nucleic acids. The invention also provides for the use of ASIC-2S.2, as well as agonists, antagonists or antibodies specifically binding ASIC-2S.2. in the diagnosis, prevention and treatment of diseases associated with expression of ASIC-2S.2. Also are disclosed methods of influencing electrophysiological, pharmacological and/or functional properties of ASIC-2S.2 as well as methods for screening for substances having ion-channel modulating activity or substances capable of disrupting subunit association or interaction.

FIELD OF THE INVENTION

[0001] The present invention is based on the discovery of a novel AcidSensing Ion Channel (ASIC) with distinctive channel properties andbiological activity. This novel channel, ASIC-2S.2 is a heteromultimericcomplex comprised of two different types of ASIC subunits, namely ASIC2Aand ASIC3. The present invention is also based on the discovery of a newuse of polynucleotides encoding ASIC2A and ASIC3, said use being theinclusion of ASIC2A and ASIC3 in the assembly of a heteromultimeric ionchannel. This invention further includes the use of the abovecompositions for diagnosis, prevention, or treatment of diseases relatedto the expression of the heteromultimeric ASIC channel disclosed herein.

[0002] The present invention demonstrates for the first time the directbiochemical interaction between two distinct ASIC subunits, namelyASIC2A and ASIC3, which produces a novel proton-gated ion channel withdistinctive properties.

BACKGROUND OF INVENTION

[0003] In mammals, the pH of the extracellular compartment, includinginterstitial fluids and blood, is strictly regulated and maintained at aconstant value of 7.4. Acid sensing is a specific kind of chemoreceptionthat plays a critical role in the detection of nociceptive pH imbalancesoccurring, for example, in conditions of cramps, trauma, inflammation orhypoxia (Lindahl, Adv Neurol 1974; 4: 45)). In mammals, a population ofsmall-diameter primary sensory neurons in the dorsal root ganglia andtrigeminal ganglia (Bevan and Yeats, J Physiol (Lond) 1991; 433: 145) aswell as central neurons (Varming, Neuropharmacol 1999; 38: 1875) expressspecialized pH-sensitive surface receptors activated by an increase ofextracellular proton concentrations. Acid sensitivity of sensory as wellas central neurons is mediated by a family of proton-gated cationchannels structurally related to C. elegans degenerins (DEG) andmammalian epithelial sodium channels (ENaC). This invention relates tothese Acid Sensing Ion Channels (ASIC) and specifically reports thediscovery of novel class of receptors generated by the heteromultimericassembly of two distinct ASIC subunits, namely ASIC2A (or BnaC1, orBNC1, or MDEG, or MDEG1) and ASIC3 (or hASIC3, or DRASIC) and usesthereof.

[0004] Tissue acidosis is associated with a number of painful,physiological (e.g. cramps) and pathological conditions (e.g.inflammation, intermittent claudication, myocardial infarction).Experimentally, similar painful events can be reproduced by infusing lowpH solutions into skin or muscle. Furthermore, the prolonged intradermalinfusion of low pH solutions can mimic the characteristic hyperalgesiaof chronic pain. To further characterize the effects of protons andtheir relation to pain, low pH solutions were applied to patch-clampedcentral and peripheral sensory neurons. Inward currents were inducedwhen pH was dropped to acidic values, providing evidence for theexistence of proton-activated ion channels. Several types of nativecurrents were observed in sensory neurons from rat and human trigeminaland dorsal root ganglia as well as central neurons: rapidly inactivatingcurrents; non-inactivating currents; and biphasic currents displaying arapidly inactivating current followed by non-inactivating sustainedcurrent. Other differences regarding ion selectivities were alsoreported. These results suggested the existence of a multigene family ofproton-gated ion channels, implicated in neurotransmission and/orneuromodulation.

[0005] Cloned Proton-Gated Ion Channels

[0006] The mammalian proton-gated cation channels have recently beencloned and named <<ASIC>> for Acid Sensing Ion Channels. Sequenceanalysis identifies them as members of the DEG/ENaC superfamily of ionchannels. The putative membrane topology of ASIC receptors predicts twotransmembrane spanning domains with both N- and C-termini in theintracellular compartment, as shown for the epithelial sodium channels.Four sub-classes of ASIC receptors have been identified:

[0007] 1. ASIC1 ion channels display rapidly inactivating inwardcurrents (Waldmann et al., Nature 1997; 386:173)

[0008] 2. ASIC2 ion channels display slowly inactivating inward currents(Brassilana et al., J Biol Chem 1997; 272: 28819).

[0009] 3. ASIC3 ion channels display biphasic inward currents with aninitial rapidly inactivating component, followed by a sustainednon-inactivating current (Waldmann et al., J Biol Chem 1997; 272: 20975;Babinski et al., J Neurochem 1999; 72: 51)

[0010] 4. ASIC4 ion channels displaying similar properties as those ofASIC3 (Wood et al., WO9963081)

[0011] Other recently discovered ion channel subunits, BLINaC and INaC,appear to belong to the ASIC family but are not activated by protons andhave not yet been shown to interact with other ASIC subunits (Sakai etal., J Physiol 1999; 519: 323, Schaefer et al., FEBS Lett 2000; InPress).

[0012] Families of ASIC Receptors Created by Alternative Splicing ofmRNAs

[0013] A common feature of these ion channels is the existence ofalternative splice variants, which display important functionaldifferences. Indeed, the replacement of the first 185 amino acids ofASIC1 (hereinafter named ASIC1A) by a distinct new sequence of 172 aminoacids generates a new channel, ASIC1B, which has similar currentkinetics as ASIC1A but needs lower pH values for activation (pH₅₀ of 6.2and 4.5, respectively, for ASIC1A and ASIC1B). Also, it appears thatASIC1B is specifically expressed in rat dorsal root ganglia. A similarsituation is also observed with rat ASIC2 (hereinafter named ASIC2A),where the replacement of the first 185 amino acids by a distinct newsequence of 236 amino acids generates another ASIC ion channel subunit,ASIC2B. When expressed alone as a homomultimer in mammalian cells orXenopus oocytes, ASIC2B does not appear to be activated by low pHsolutions. ASIC3, which has been identified in human, also appears toexist in various forms. Indeed, DRASIC is an ASIC3-like channelidentified in rat, which displays 85% identity with the human ASIC3sequence and has similar biphasic current kinetics. However, importantdifferences regarding tissue distribution, ion selectivities and pH₅₀suggest that DRASIC might not be the human orthologue of ASIC3 butrather a different subtype. Furthermore, the existence of two 3′ splicevariants of human ASIC3 (ASIC3B and 3C, sequences submitted to GenBank)have been reported but differences in function have yet to bedocumented. Alternative splicing, therefore, appears like an importantmechanism for increasing the diversity of ASIC receptors, which mostprobably assume critical roles in the nervous system, such asneurotransmission, nociception or mechanosensation (see below).

[0014] Families of ASIC Receptors Created by HeteromultemericAssociations

[0015] In general, functional ion channels are complex structurescomprised of several individual components, referred) to as subunits.The number of subunits depends on the type of ion channel and subunitscan either be all identical (homomultimeric channels) or include acombination of several different subtypes (heteromultimeric channels).For example, Epithelial sodium Channels (ENaC), which belong to the samegene family as ASIC receptors, are comprised of at least three differentsubunits, namely αEnaC, βEnaC and γEnaC (Canessa et al., Nature 1994;367: 463). Although cloned ASIC receptors have mostly been characterizedin vitro in their homomultimeric form, the analogy with EnaCs raises thepossibilty that ASIC subunits might also associate in variouscombinations to generate novel channels with distinctive properties.Indeed, heteromultimeric ASIC channels might account for some of thenative proton-gated currents still not explained by any of thehomomultimeric ASICs cloned to date. Examples of such native currentsare the sustained non-desensitizing currents seen at pH 6 (Bevan andYeats, J Physiol 1991; 433: 145). Furthermore, the discovery of theproton-insensitive ASIC2B (or MDEG2) suggests that it may function as anaccessory subunit. Indeed, the first evidence for heteromultimeric ASICreceptors came from coexpression studies featuring rat ASIC2B eitherwith ASIC2A or with ASIC3. Channels created by ASIC2A and ASIC2B appearto be slightly more sensitive to pH, while inward currents carried byASIC2B+ASIC3 channels are apparently less sodium selective than thehomomultimeric ASIC3 currents (Lingueglia et al., J Biol Chem 1997: 272:29778). However, no biochemical evidence of interaction has beenreported to date for any ASIC subunits. Furthermore, other coexpressionexperiments with different subunits suggest that not all subunitcombinations yield novel functional channels. Thus, the composition andfunctional characteristics of heteromultimeric ASIC channels aretherefore unpredictable.

SUMMARY OF THE INVENTION

[0016] It is the purpose of the present invention to disclose anddescribe a novel heteromultimeric ASIC channel, herein called ASIC-2S.2,and uses thereof.

[0017] The present invention reports the discovery of a novelheteromultimeric ASIC receptor (hereinafter called ASIC-2S.2). Alsocontemplated within the scope of this invention is the potentialinvolvement of this new receptor in neurotransmission and/or nociceptionand/or mechanosensation and/or any other neurological and/or metabolicprocesses in normal and/or pathophysiological conditions. This inventionseeks also to cover any uses of this new ion channel as a therapeutictarget, including but not limited to drug screening technologies (i.e.screening for channel antagonists, agonists, modulators and/or subunitassociation blockers), diagnostic marker, or gene therapies. Also withinthe scope of the present invention is the heteropolymerization of theASIC-2S.2 channel with one or more additional subunits of the ASICfamily from any species, including but not limited to ASIC1, ASIC1A,BNaC2, ASIC1B, ASIC2B, MDEG2, ASIC4, SPASIC or any variants thereof, aswell as heteropolymerization of ASIC-2S.2 with any other members of theDegenerin and EnaC family from any species.

[0018] An object of this invention is therefore to provide thecomposition of the novel ASIC-2S.2 receptor and methods of producing andexpressing functional ASIC-2S.2 ion channels.

[0019] Another object of the present invention is to provide methods forengineering nucleic acids specifically designed to encode a chimericASIC receptor comprised of a single polypeptide, where two or more ASICsubunits are covalently linked together, and expressed in tandem as asingle amino acid sequence.

[0020] Also included within the scope of this invention are methodsdesigned for screening and identifying substances, whether chemicallysynthetised or isolated from natural sources, which have ion channelmodulating activity. Typically this includes but is not limited tocompetitive and non-competitive agonists and partial agonists as well ascompetitive and non-competitive antagonists and partial antagonists, aswell any substance capable of directly or indirectly disrupting,inhibiting or preventing the complete or partial association of ASICsubunits, as disclosed hereinafter, into functional, partiallyfunctional, or non-functional channels.

[0021] The invention additionally features the specific use of nucleicacids encoding the ASIC subunits comprising the ASIC-2S.2heteromultimeric channel, namely ASIC2A (BNaC1, MDEG1) and ASIC3 (orDRASIC), as well as polypeptides, oligonucleotides, peptide nucleicacids (PNA), fragments, portions, antisense molecules, or anyderivatives thereof, where specific use includes disruption, inhibitionor prevention of ASIC subunit association or assembly into the ASIC-2S.2heteromultimeric channels of the present invention. Also within thescope of this invention are expression vectors and host cells comprisingnucleic acids that simultaneously encode and/or express ASIC2A and ASIC3subunits together. The present invention also features pharmaceuticalcompositions comprising substantially purified ASIC-2S.2 as well asantibodies which bind specifically to the ASIC subunits of the ASIC-2S.2channel complex and whose specific binding causes the disruption,inhibition or prevention of ASIC subunit association or assembly intothe ASIC-2S.2 heteromultimeric channels of the present invention

DESCRIPTION OF THE FIGURES

[0022] The following drawings, figures and tables are illustrative ofthe embodiments of the invention and are not meant to limit the scope ofthe invention as encompassed by the claims.

[0023]FIG. 1 illustrates the pH-activated inward currents recorded involtage clamped Xenopus oocytes expressing ASIC2A and ASIC3, eitheralone or in combination. The functional interaction is clearly visiblewhen comparing currents between mono-injected and co-injected oocytes.FIG. 1A: Human subunits, BNaC1 and hASIC3; FIG. 1B: Rat subunits, MDEG1and DRASIC.

[0024]FIG. 2 shows the pH dose-response curves of proton-induced inwardcurrents in human and rat homomultimeric (ASIC2A or ASIC3) andheteromultimeric (ASIC-2S.2)-expressing oocytes.

[0025]FIG. 3 shows the antagonistic effects of amiloride (A) andgadolinium ions (B) on proton-induced inward currents in human and rathomomultimeric (ASIC2A or ASIC3) and heteromultimeric(ASIC-2S.2)-expressing oocytes.

[0026]FIG. 4 represents an ethidium bromide stained agarose gel showingthe expression pattern of mRNA for hASIC2A and hASIC3 determined byduplex RT-PCR amplification of commercially available human RT-cDNA(Clontech) using specific oligonucleotide primers for each subunit.Noteworthy is the co-expression of both subunits in trigeminal ganglia,suggesting that the heteromultimeric receptor, ASIC-2S.2 might beinvolved in pain and/or sensory transmission.

[0027]FIG. 5 reveals In situ hybridization in rat cerebellum with MDEG1-and DRASIC-specific probes showing the co-expression of both subunits inthe same cell type.

[0028]FIG. 6 illustrates the effects of N- or C-terminal tagging ofASIC2A and/or ASIC3 subunits on proton-activated inward currentsrecorded using voltage clamped Xenopus oocytes expressing either thehomomultimeric or heteromultimeric ASIC channels. FIG. 6A gives examplesof currents and FIG. 6B summarises results in a table format.

[0029]FIG. 7 is a Western blot of N-terminally tagged hASIC2A and hASIC3subunits coexpressed in Xenopus oocytes (A) or HEK293 cells (B), showingthat both subunits are co-immunoprecipitated or co-purified, providingevidence in favour of a direct biochemical interaction between subunits.

[0030]FIG. 8 is a Table listing all the synonyms of ASIC2A and ASIC3used in various publications, databases, patent applications andpatents.

[0031]FIG. 9 shows the inhibitory effects of C-terminal and N-terminalfragments of ASIC2A or ASIC3 on the current mediated by ASIC2S.2heteromoeric channel. Any given vectors carrying the fragments wasco-injected with the ASIC2A and ASIC3 subunits and tested at twodifferent ratios of fragment versus ASIC2A and ASIC3 subunit. FIG. 9Ashows the effects of the C-terminal fragments of ASIC3 (upper traces)and ASIC2A (lower traces). FIG. 9B shows the effects of the N-terminalfragments of ASIC3 (upper traces) and ASIC2A (lower traces).

[0032]FIG. 10 reveals the selective modulatory effect of 10 μM NPFF onthe proton-gated cationic currents evoked by human heteromeric ASICreceptors expressed in Xenopus oocytes. A. Human homomeric ASIC2A, ASIC3and heteromeric ASIC2S.2 (ASIC2A+3) response to pH 4 application (bar).B. Same as in A but in the presence of NPFF. Note the change indesensitization kinetics in presence of the peptide and the potentiationof the current mediated by heteromeric ASIC2S.2. C. Quantitative effectsof NPFF on peak currents evoked by acidic pH for the three subtypes ofhuman ASICs. Values expressed as % of control are mean±SEM

[0033]FIG. 11 shows the modulatory effect of 10 μM NPFF on theproton-gated cationic currents evoked by rat homomeric ASIC3 andheteromeric ASIC2S.2 (ASIC2A+3) receptors expressed in Xenopus oocytes.A. rat homomeric ASIC2A, ASIC3 and heteromeric ASIC2S.2 response to pH 4application (bar). B. Same as in A but in the presence of NPFF. Note themore pronounced potentiation of the current mediated by heteromericASIC2S.2 than by homomeric ASIC3. C. Histograms illustrating the effectsof NPFF on peak currents evoked by acidic pH for the three subtypes ofrat ASICs. Values expressed as % of control are mean±SEM.

[0034]FIG. 12 Dose-response curves of NPFF (A) and FMRFamide (B) on thepotentiation of currents induced by pH 4 on human ASIC2S.2 (ASIC2A+3)receptors. NPFF was found more potent (EC50=2 μM, n=6) than FMRFamide(EC50=13 μM, n=12) in modulating ASIC2S.2-mediated cation currents.Values are expressed as mean±SEM and represent % of response to pH 4measured in presence of 10-8 M peptide.

[0035]FIG. 13A. NPFF potentiated the response of human ASIC2S.2(ASIC2A+3) receptors to pH 6 stimulation. B. pH dose-response curves ofhuman heteromeric ASIC2S.2 (ASIC2A+3) receptors in the presence orabsence of neuropeptides. Sensitivity and maximal response toacidification increased in presence of 100 μM FMRFamide and the effectswere even greater in the presence of 10 μM NPFF. Reference value forcurrent normalization corresponded to maximal control currents inducedat pH 3. Values are expressed as mean±SEM.

DETAILED DESCRIPTION OF THE INVENTION

[0036] Preambule

[0037] It is understood that the present invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0038] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” includes a plurality of such host cells; reference to “theantibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

[0039] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

[0040] Definitions

[0041] For the sake of clarity, FIG. 8 summarises the nomenclature ofthe ASIC subunits encompassed by the present invention and will beapplied to identify without ambiguity the ASIC subunits herein referredto. Unless specified otherwise, reference to an ASIC subunit is notintended to be limited to a particular species but includes theorthologues or homologues of any species.

[0042] “Polynucleotide” and “nucleic acids” as used herein refers tosingle- or double-stranded molecules which may be “deoxyribonucleicacid” (DNA), comprised of the nucleotide bases A, T, C and G, or“ribonucleic acid” (RNA), comprised of bases A, U (substitutes for T), Cand G. Polynucleotides may represent a coding strand or its complement,the sense or anti-sense strands. Polynucleotides may be identical insequence to the sequence which is naturally occurring or may includealternative codons which encode the same amino acid as that which isfound in the naturally occurring sequence (Lewin: “Genes V”, Chapter 7;Oxford University Press, 1994). Furthermore, polynucleotides may includecodons which represent conservative substitutions of amino acids. Theterm “polynucleotide” will also include all possible alternate forms ofDNA or RNA, such as genomic DNA (both introns and exons), complementaryDNA (cDNA), cRNA, messenger RNA (mRNA), and DNA or RNA prepared bypartial or total chemical synthesis from nucleotide bases, includingmodified bases, such as tritylated bases and unusual bases such asinosine. Polynucleotides will also embrace all chemically, enzymaticallyor metabolically modified forms of DNA or RNA, as well as the chemicalforms of DNA and RNA characteristic of viruses.

[0043] The term “oligonucleotide” or “oligo” will refer shortpolynucleotides, typically between 10 to 40 bases in length.

[0044] “Polypeptide” as used herein refers to a molecule comprised oftwo or more amino acids joined to each other by peptide bonds ormodified peptide bonds (i.e. isosteres). Amino acids include all 20naturally gene-encoded amino acids as well as naturally or chemicallymodified amino acids. Polypeptides refer to both short chains of aminoacids, commonly referred to as peptides, oligopeptides, or oligomers,and to longer chains, commonly referred to as proteins. Thus, “aminoacid sequence” as used herein refers to an oligopeptide, peptide,polypeptide, or protein molecule and fragments or portions thereof,corresponding to a naturally occurring or synthetic molecule. Where“amino acid sequence” is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, “amino acidsequence” and like terms, such as “polypeptide” or “protein” are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule. Furthermore,polypeptides will also include amino acid sequences modified either bynatural processes, such as posttranslational processing, or by chemicalmodification techniques, which are well known in the art. A givenpolypeptide may contain many types of modifications or a givenmodification may be present in the same or varying degrees at severalsites in a given polypeptide. Modifications can occur anywhere in thepolypeptide, including but not limited to, the peptide backbone, theamino acid side-chains and the amino or carboxyl termini. All the abovereferred to modifications as well as their practice are well describedin the research literature, both in basic texts and detailed monographs(“Proteins: Structure and Molecular Properties”; Creighton T E, FreemanW H, 2^(nd) Ed., New-York, 1993; “Posttranslational CovalentModification of Proteins”, Johnson B C, ed., Academic Press, New-York,1983; Also: Seiter et al., Meth Enzymol 1990; 182: 626, and Rattan etal., Ann NY Acad Sci 1992; 663: 48).

[0045] “Peptide nucleic acid”, as used herein, refers to a moleculewhich comprises an oligonucleotide to which an amino acid residue, suchas lysine, and an amino group have been added. These small molecules,also designated anti-gene agents, stop transcript elongation by bindingto their complementary strand of nucleic acid (Nielsen et al. AnticancerDrug Des 1993; 8: 53).

[0046] ASIC2A or ASIC3, as used herein, refers to the amino acidsequences of substantially purified ASIC2A and ASIC3 obtained preferablybut not exclusively from human or rat, from any source whether natural,synthetic, semi-synthetic, or recombinant.

[0047] The term “variant” as used herein is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptide,respectively. A typical variant of a polynucleotide differs innucleotide sequence from another reference polynucleotide. Differencesin the nucleotide sequence of the variant may or may not alter the aminoacid sequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,insertions, deletions, fusions, and truncations in the polypeptideencoded by the reference sequence, as discussed below. A typical variantof a polypeptide differs in amino acid sequence from another referencepolypeptide. Generally, differences are such that the sequences of thereference polypeptide and the variant are closely similar overall and,in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,insertions or deletions in any combination. A substituted or insertedamino acid residue may or may not be one encoded by the genetic code. Avariant of a polynucleotide or polypeptide may be naturally occurringsuch as an allelic or a pseudoallelic variant, including polymorphismsor mutations at on or more bases, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniquesor by direct synthesis. The term “mutant” are encompassed by the abovedefinition of non-natural variants.

[0048] “splice variants” as referred to herein are variants, whichresult from the differential or alternative splicing and assembly ofexons present in a given gene. Typically, the encoded proteins willdisplay total identity in most regions, but lower identity in thespecific region encoded by different exons.

[0049] A “deletion”, as used herein, refers to a change in either aminoacid or nucleotide sequence in which one or more amino acids ornucleotide residues, respectively, are absent, as compared to areference polypeptide or polynucleotide.

[0050] An “insertion” or “addition”, as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid or nucleotide residues, respectively, as compared toa reference polypeptide or polynucleotide.

[0051] A “substitution”, as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively, as compared to a reference polypeptide orpolynucleotide.

[0052] The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding ASIC2A or ASIC3 or the encodedASIC2A or ASIC3. Illustrative of such modifications would be replacementof hydrogen by an alkyl, acyl, or amino group. A nucleic acid derivativewould encode a polypeptide which may or may not retain some or all ofthe essential biological characteristics of the natural molecule.

[0053] The term “identity” as used herein refers to a measure of theextent of identical nucleotides or amino acids that two or morepolynucleotide or amino acid sequences have in common. In general, thesequences are aligned so that the highest order match is obtained,referred to as the “alignment”. Such optimal alignments make use ofgaps, which are inserted to maximize the number of matches using localhomology algorithms, such as the Smith-Waterman alignment. The terms“identity”, or “similarity”, or “homology”, or “alignment” are wellknown to skilled artisans and methods to perform alignments and measureidentity are widely described and taught in the literature: Dayhoff etal., Meth Enzymol 1983; 91: 524—Lipman D J and Pearson W R, Science1985; 227: 1435—Altschul et al., J Mol Biol 1990; 215: 403.—Pearson W R,Genomics 1991; 11: 635.—Gribskov M and Devreux J, eds. (1992) SequenceAnalysis Primer, W H Freeman & Cie, New-York.—Altschul et al., NatureGen 1994; 6: 119. Furthermore, methods to perform alignments and todetermine identity and similarity are codified in computer programs andsoftware packages, some of which may also be web-based and accessible onthe internet. Preferred software include but are not limited to BLAST(Basic Local Alignment Search Tools), including Blastn, Blastp, Blastx,tBlastn (Altschul et al., J Mol Biol 1990; 215: 403), FastA and TfastA(Pearson and Lipman, PNAS 1988; 85: 2444), Lasergene99 (DNASTAR, MadisonWis.), Omiga 2.0 or MacVector (Oxford Molecular Group, Cambridge, UK),Wisconsin Package (Genetic Computer Group (GCG), Madison, Wis.), VectorNTI Suite (InforMax Inc., N. Bethesta, Md.), GeneJockey II (Biosoft,Cambridge, UK).

[0054] As an illustration, by a polynucleotide having a nucleotidesequence with at least, for example, 95% “identity” to a referencenucleotide sequence of SEQ ID NO: 1, is intended that the nucleotidesequence of the polynucleotide is identical to the reference sequenceexcept that the polynucleotide sequence may include up to five pointmutations, or divergent nucleotides, per 100 nucleotides of thereference nucleotide sequence of SEQ ID NO: 1. In other words, to obtaina polynucleotide having a nucleotide sequence at least 95% identical toa reference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence.These mutations of the reference sequence may occur at the 5′ or 3′terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more continuousgroups within the reference sequence.

[0055] Similarly, by a polypeptide having an amino acid sequence havingat least, for example, 95% “identity” to a reference amino acid sequenceof SEQ ID NO: 2, is intended that the amino acid sequece of thepolypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid sequence of SEQ ID NO:2. In other words, to obtain a polypeptide having an amino acid sequenceat least 95% identical to a reference amino acid sequence, up to 5% ofthe amino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence, or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more continuous groups within thereference sequence.

[0056] The term “biologically active” or “biological activity”, as usedherein, refer to a protein having structural, regulatory, biochemical,electrophysiological or cellular functions of a naturally occurringmolecule. Likewise, “immunologically active” refers to the capability ofthe natural, recombinant, or synthetic ASIC2A, or ASIC3, or ASIC-2S.2,or any oligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0057] As used herein, “specific antibodies” “antibodies to ASIC-2S.2”and “ASIC-2S.2-specific antibodies” refer to antibodies whichspecifically bind the ASIC-2S.2 protein complex, or specifically bindASIC2A and/or ASIC3 in their heteromultimeric conformation, orantibodies specifically binding ASIC2A or ASIC3 and capable ofinhibiting, preventing or disrupting the interaction, association orassembly between ASIC2A and ASIC3, as described herein.

[0058] As used herein, “proton-gated” and “acid-sensing” refer to anincrease in cation permeability of a channel molecule induced by anincrease in proton ion concentration, also described as increasedacidity or lowering of pH.

[0059] As used herein, “gain of function” refers to a potentiation of anexisting biological activity and/or an acquisition of a novel biologicalactivity. Similarly, “Loss of function” refers to a partial or completeloss of one or more existing biological activities. The expression“Dominant-negative” refers to a loss-of-function derivative of ASIC2A orASIC3, which when coexpressed with a fully functional ASIC2A or ASIC3,in vivo, for example as a transgene, or in vitro, for example in anassay used to test the specific biological activity (for example“acid-sensing”), will dominate the response and impose the loss ofbiological activity on all other ASIC subunits associated with it.

[0060] The term “agonist”, as used herein, refers to a molecule, whichcauses a change in ASIC-2S.2 and modulates or induces directly orindirectly a biological activity of the ASIC-2S.2 heteromultimericchannels. Agonists may include proteins, nucleic acids, aptamers,carbohydrates, or any other molecules, which display the propertiesdescribed herein above.

[0061] The terms “antagonist” or “inhibitor”, as used herein, refer to amolecule which modulates or blocks directly or indirectly a biologicalactivity of the ASIC-2S.2 heteromultimeric channels. Antagonists andinhibitors may include proteins, nucleic acids, aptamers, carbohydrates,or any other molecules which display the properties described hereinabove.

[0062] The term “modulate”, as used herein, refers to a change or analteration in the biological activity of ASIC-2S.2 heteromultimericchannels. Modulation may be an increase or a decrease in proteinactivity, a change in binding characteristics, or any other change inthe biological, functional or immunological properties of the ASIC-2S.2.

[0063] The term “mimetic”, as used herein, refers to a molecule, thestructure of which is developed from knowledge of the structure ofASIC2A or ASIC3 or portions thereof and, as such, is able to effect someor all of the actions of ASIC-like molecules.

[0064] The term “substantially purified”, as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, more preferably 90%, even more preferable 95%, andmost preferably 99% free from other components with which they arenaturally associated.

[0065] “Amplification” as used herein refers to the production ofadditional copies of a nucleic acid and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art (“PCRPrimer: a laboratory manual” Dieffenbach C W and Dveksler G S, eds.,1995, CSHL Press, Plainview, N.Y.).

[0066] The term “hybridization”, as used herein, refers to any processby which a strand of nucleic acid binds with a complementary strandthrough base pairing.

[0067] The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., RNAse ProtectionAssay analysis) or between one nucleic acid sequence present in solutionand another nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

[0068] The terms “complementary” or “complementarity”, as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids' bind, or it may be complete when totalcomplementarity exists between the single-stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acid strands.

[0069] The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

[0070] As known in the art, numerous equivalent conditions may beemployed to comprise either low or high stringency conditions. Factorssuch as the length and nature (DNA, RNA, base composition) of thesequence, nature of the target (DNA, RNA, base composition, presence insolution or immobilization, etc.), and the concentration of the saltsand other components (e.g., the presence or absence of formamide,dextran sulfate and/or polyethylene glycol) are considered and thehybridization solution may be varied to generate conditions of eitherlow or high stringency different from, but equivalent to, the abovelisted conditions.

[0071] The term “stringent conditions”, as used herein, is the“stringency” which occurs within a range from about Tm-5° C. (5° C.below the melting temperature (Tm) of the probe) to about 20° C. to 25°C. below Tm. As will be understood by those of skill in the art, thestringency of hybridization may be altered in order to identify ordetect identical or related polynucleotide sequences.

[0072] The term “antisense”, as used herein, refers to nucleotidesequences which are complementary to a specific DNA or RNA sequence. Theterm “antisense strand” is used in reference to a nucleic acid strandthat is complementary to the “sense” strand. Antisense molecules may beproduced by any method, including synthesis by ligating the gene(s) ofinterest in a reverse orientation to a viral promoter, which permits thesynthesis of a complementary strand. Once introduced into a cell, thistranscribed strand combines with natural sequences produced by the cellto form duplexes. These duplexes then block either the furthertranscription or translation. In this manner, mutant phenotypes may begenerated. The designation “negative” is sometimes used in reference tothe antisense strand, and “positive” is sometimes used in reference tothe sense strand.

[0073] The term “portion”, as used herein, with regard to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from four amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ IDNO:2” encompasses the full-length human ASIC2A and fragments thereof.

[0074] “Transformation” or “transfection”, as defined herein, describesa process by which exogenous DNA enters and changes a recipient cell. Itmay occur under natural or artificial conditions using various methodswell known in the art. Transformation or transfection may rely on anyknown method for the insertion of foreign nucleic acid sequences into aprokaryotic or eukaryotic host cell. The method is selected based on thehost cell being transformed and may include, but is not limited to,viral infection, electroporation, lipofection, and particle bombardment.Such “transformed” or “transfected” cells include stably transformedcells in which the inserted DNA is capable of replication either as anautonomously replicating plasmid or as part of the host chromosome. Theyalso include cells that transiently express the inserted DNA or RNA forlimited periods of time.

[0075] The term “antigenic determinant”, as used herein, refers to thatportion of a molecule that makes contact with a particular antibody(i.e., an epitope). When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0076] The terms “specific binding” or “specifically binding”, as usedherein, in reference to the interaction of an antibody and a protein orpeptide, mean that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words, the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A”, the presence of aprotein containing epitope A (or free, unlabeled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

[0077] The term “sample”, as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acids encodingASIC2A and ASIC3 or fragments thereof may comprise a cell, chromosomesisolated from a cell (e.g., a spread of metaphase chromosomes), genomicDNA (in solution or bound to a solid support such as for Southernanalysis), RNA (in solution or bound to a solid support such as fornorthern analysis), cDNA (in solution or bound to a solid support), anextract from cells or a tissue, and the like.

[0078] The term “correlates with expression of a polynucleotide”, asused herein, indicates that the detection by northern analysis and/orRT-PCR of the presence of ribonucleic acid that is related to SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 is indicative of thepresence of mRNA encoding, respectively, hASIC2A, hASIC3, rASIC2A orrASIC3 in a sample and thereby correlates with expression of thetranscript encoding the protein.

[0079] “Alterations” in the polynucleotides of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5 or SEQ ID NO:7, as used herein, comprise any alteration inthe sequence of polynucleotides encoding, respectively, hASIC2A, hASIC3,rASIC2A or rASIC3, including deletions, insertions, and point mutationsthat may be detected using hybridization assays. Included within thisdefinition is the detection of alterations to the genomic DNA sequencewhich encodes hASIC2A, hASIC3, rASIC2A or rASIC3 (e.g., by alterationsin the pattern of restriction fragment length polymorphisms capable ofhybridizing to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7),the inability of a selected fragment of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5 or SEQ ID NO:7 to hybridize to a sample of genomic DNA (e.g., usingallele-specific oligonucleotide probes), and improper or unexpectedhybridization, such as hybridization to a locus other than the normalchromosomal locus for the polynucleotide sequence encoding hASIC2A,hASIC3, rASIC2A or rASIC3 (e.g., using fluorescent in situ hybridization(FISH) to metaphase chromosome spreads).

[0080] As used herein, the term “antibody” refers to intact molecules aswell as fragments thereof, such as Fa, F(ab′)₂, and Fv, which arecapable of binding the epitopic determinant. Antibodies that bindhASIC2A, hASIC3, rASIC2A or rASIC3 polypeptides can be prepared usingintact polypeptides or fragments containing small peptides of interestas the immunizing antigen. The polypeptide or peptide used to immunizean animal can be derived from translated RNA or synthesized chemically,and can be conjugated to a carrier protein, if desired. Commonly usedcarriers that are chemically coupled to peptides include bovine serumalbumin and thyroglobulin. The coupled peptide is then used to immunizethe animal (e.g., a mouse, a rat or a rabbit). These methods are welldescribed in the literature: e.g. “Antobodies: A Laboratory Manual”,Harlow E and Lane D, eds., 1998, CSHL Press, Plainview, N.Y.).

[0081] The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

DISCLOSURE OF THE INVENTION

[0082] The present invention is based on the discovery of a novel AcidSensing Ion Channel (ASIC) with distinctive channel properties andbiological activity. This novel channel, ASIC-2S.2 is a heteromultimericcomplex comprised of two different types of ASIC subunits, namely ASIC2Aand ASIC3 (see FIG. 8). The present invention is also based on thediscovery of a new use of polynucleotides encoding ASIC2A and ASIC3,said use being the inclusion of ASIC2A and ASIC3 in the assembly of aheteromultimeric ion channel. This invention further includes the use ofthe above compositions for diagnosis, prevention, or treatment ofdiseases related to the expression of the heteromultimeric ASIC channeldisclosed herein. The preferred polynucleotides are those of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7, encoding, respectively,the hASIC2A, hASIC3, rASIC2A and rASIC3 of SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6 and SEQ ID NO:8. The above enumerated polynucleotides havealready been embodied in the following patents or patent applications:U.S. Pat. No. 5,892,018; WO9835034; WO9921981; WO9854316. The generalnotion of heteromultimeric associations between subunits of the samefamily is widely recognized by those skilled in the art. The inventivestep resides in finding the functional combinations of known subunits aswell as the changes in channel properties and/or biological activity.Patent application WO9835034 does indeed claim hybrid ASIC channels butsupports this claim with indirect evidence suggesting interactionsbetween ASIC and MDEG1, MDEG1 and MDEG2, and between MDEG2 and DRASIC(see above). The present invention demonstrates for the first time thedirect biochemical interaction between two distinct ASIC subunits,namely ASIC2A and ASIC3, which produces a novel proton-gated ion channelwith distinctive properties.

[0083] The invention also encompasses heteromultimeric ASIC channelscomprised of ASIC2A and/or ASIC3 variants, in any possible combinationof wild-type and variant forms. A preferred ASIC2A variant is one havingat least 80%, and more preferably 90%, amino acid sequence identity tothe ASIC2A amino acid sequence of SEQ ID NO: 2 or SEQ ID NO.6. A mostpreferred ASIC2A variant is one having at least 95% amino acid sequenceidentity to SEQ ID NO: 2 or SEQ ID NO:6, while those with 97-99% aminoacid sequence identity are most highly preferred. A preferred ASIC3variant is one having at least 80%, and more preferably 90%, amino acidsequence identity to the ASIC3 amino acid sequence of SEQ ID NO:4 or SEQID NO.8. A most preferred ASIC3 variant is one having at least 95% aminoacid sequence identity to SEQ ID NO:4 or SEQ ID NO:8, while those with97-99% amino acid sequence identity are most highly preferred.

[0084] The invention also encompasses polynucleotides which encodeASIC2A or ASIC3 polypeptides. Accordingly, any nucleic acid sequence,which encodes the amino acid sequence of ASIC2A or ASIC3 can be used togenerate recombinant molecules which express ASIC2A or ASIC3. In aparticular embodiment, the invention encompasses the polynucleotidecomprising the nucleic acid of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5,and SEQ ID NO:7.

[0085] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding ASIC2A or ASIC3, some bearing minimal homology to thenucleotide sequences of any known and naturally occurring gene, may beproduced. Thus, the invention contemplates each and every possiblevariation of nucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe nucleotide sequence of naturally occurring ASIC2A or ASIC3, and allsuch variations are to be considered as being specifically encompassed.

[0086] Although nucleotide sequences which encode ASIC2A or ASIC3 andtheir variants are preferably capable of hybridizing to the nucleotidesequence of the naturally occurring ASIC2A or ASIC3 under appropriatelyselected conditions of stringency, it may be advantageous to producenucleotide sequences encoding ASIC2A or ASIC3 or their derivativespossessing a substantially different or non-naturally occurring codonusage. Codons may be selected to increase the rate at which expressionof the peptide occurs in a particular prokaryotic or eukaryoticexpression host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding ASIC2A or ASIC3 and theirderivatives without altering the encoded amino acid sequences includethe production of RNA transcripts having more desirable properties, suchas a greater half-life, than transcripts produced from the naturallyoccurring sequence.

[0087] The invention also encompasses production of a DNA sequence, orportions thereof, which encode ASIC2A or ASIC3 and their derivatives,entirely by synthetic chemistry. After production, the synthetic genemay be inserted into any of the many available DNA vectors and cellsystems using reagents that are well known in the art at the time of thefiling of this application. Moreover, synthetic chemistry may be used tointroduce mutations into a sequence encoding ASIC2A or ASIC3 or anyportion thereof.

[0088] Further encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed nucleotide sequences, andin particular, to those shown in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, under various conditions of stringency.Hybridization conditions are based on the melting temperature (Tm) ofthe nucleic acid binding complex or probe, as taught in Berger andKimmel (Meth Enzymol 1987: 152), and may be used at a definedstringency. Excluded from the above defined polynucleotides arepolynucleotides disclosed in Patent Application WO9835034 under SEQ IDNO:1, SEQ ID NO:2, and SEQ ID NO:6.

[0089] Altered nucleic acid sequences encoding ASIC2A or ASIC3 which areencompassed by the invention include deletions, insertions, orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent ASIC2A or ASIC3. Theencoded protein may also contain deletions, insertions, or substitutionsof amino acid residues, which result in a functionally equivalent ASIC2Aor ASIC3. Also encompassed by the invention are altered nucleic acidsequences, including deletions, insertions or substitutions, whichresult in a polynucleotide that encodes an ASIC2A or ASIC3 polypeptidewith increased or novel biological activity (“gain of function”), or anASIC2A or ASIC3 polypeptide with decreased or suppressed biologicalactivity (“Loss of function” or “Dominant-negative”). The encodedprotein may also contain deletions, insertions, or substitutions ofamino acid residues, which result in a functionally divergent ASIC2A orASIC3, as described herein above. Deliberate amino acid substitutionsmay be made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the biological activity of ASIC2A or ASIC3 isretained. For example, negatively charged amino acids may includeaspartic acid and glutamic acid; positively charged amino acids mayinclude lysine and arginine; and amino acids with uncharged polar headgroups having similar hydrophilicity values may include leucine,isoleucine, and valine; glycine and alanine; asparagine and glutamine;serine and threonine; phenylalanine and tyrosine.

[0090] Also included within the scope of the present invention arealleles of the genes encoding ASIC2A or ASIC3. As used herein, an“allele” or “allelic sequence” is an alternative form of the gene, whichmay result from at least one mutation in the nucleic acid sequence.Alleles may result in altered mRNAs or polypeptides whose structure orfunction may or may not be altered. Any given gene may have none, one,or many allelic forms. Common mutational changes, which give rise toalleles, are generally ascribed to natural deletions, additions, orsubstitutions of nucleotides. Each of these types of changes may occuralone, or in combination with the others, one or more times in a givensequence.

[0091] Methods for DNA sequencing, which are well known and generallyavailable in the art, may be used to practice any embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, Sequenase II (US Biochemical Corp, Cleveland, Ohio),Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of recombinant polymerases andproofreading exonucleases such as the ELONGASE Amplification Systemmarketed by Gibco BRL (Gaithersburg, Md.) or the EXPAND High fidelity orLong-Template systems (Roche). Preferably, the process is automated withmachines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.),Peltier Thermal Cycler (PTC200; M.J. Research, Watertown, Mass.) and theABI 377 DNA sequencers (Perkin Elmer), to name a few.

[0092] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled devise camera. Output/light intensity may be converted toelectrical signal using appropriate software (e.g. Genotyper™ andSequence Navigator™, Perkin Elmer) and the entire process from loadingof samples to computer analysis and electronic data display may becomputer controlled. Capillary electrophoresis is especially preferablefor the sequencing of small pieces of DNA which might be present inlimited amounts in a particular sample.

[0093] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterthe ASIC2A or ASIC3 coding sequence for a variety of reasons, includingbut not limited to, alterations which modify the cloning, processing,and/or expression of the gene product. DNA shuffling by randomfragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides may be used to engineer the nucleotide sequence. Forexample, site-directed mutagenesis may be used to insert new restrictionsites, to alter glycosylation patterns, to change codon preference, toproduce splice variants, or other mutations, and so forth.Alternatively, the nucleotide sequences can be engineered to generatechimeric ASIC2A or ASIC3 channels, where portions of the ASIC2A channelare substituted with equivalent portions of the ASIC3 subunit and/orvice versa. Chimeric ASIC2A or ASIC3 can also be constructed bysubstituting portions ASIC2A or ASIC3 with equivalent portions fromother ASIC subunits, for example the ASIC1A or ASIC4. Nucleic acids canalso be engineered to encode as a single polypeptide two or more ASIC2Aand/or ASIC3 subunits in tandem.

[0094] In another embodiment of the invention, a natural, modified, orrecombinant polynucleotide encoding ASIC2A or ASIC3 may be ligated to aheterologous sequence to encode a fusion protein. For example, toprovide biochemical evidence of direct protein-protein interactionsbetween ASIC2A and ASIC3, polynucleotides encoding ASIC2A and ASIC3 aremodified to encode chimeric ASIC2A or ASIC3 proteins with N- orC-terminal extensions adding a foreign epitope recognised bycommercially available antibodies or affinity resins. A fusion proteinmay also be engineered to contain a cleavage site located between theASIC2A or ASIC3 encoding sequence and the heterologous protein sequence,so that ASIC2A or ASIC3 may be cleaved and purified away from theheterologous moiety.

[0095] In another embodiment, the coding sequence of ASIC2A and/or ASIC3may be synthesized, in whole or in part, using chemical methods wellknown in the art (see Caruthers et al., Nuc. Acids Res. Symp. Ser. 1980;215-23; Horn et al., Nuc. Acids Res. Symp. Ser. 1980; 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the ASIC2A and/or ASIC3 amino acid sequence, or a portionthereof. For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge et al., Science 1995; 269: 202) andautomated synthesis may be achieved, for example, using the ABI 431APeptide Synthesizer (Perkin Elmer).

[0096] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton T.(1983) “Proteins, Structures and Molecular Principles”, W. H. Freeman &Co., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton T (1983), supra). Additionally, theamino acid sequence of ASIC2A and/or ASIC3, or any part thereof, may bealtered during direct synthesis and/or combined using chemical methodswith sequences from other proteins, or any part thereof, to produce avariant polypeptide.

[0097] In order to express a biologically active ASIC2A and/or ASIC3,the nucleotide sequence encoding ASIC2A and/or ASIC3 or functionalequivalents thereof, may be inserted into an appropriate expressionvector, i.e., a vector, which contains the necessary elements for thetranscription and translation of the inserted coding sequence.

[0098] Methods which are well known to those skilled in the art may beused to construct expression vectors containing a ASIC2A and/or ASIC3coding sequences and appropriate transcriptional and translationalcontrol elements. These methods include in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.Such techniques are described in “Molecular Cloning: A LaboratoryManual”, Sambrook J, Ed., CSHL Press, 1989, Cold Spring Harbor, N.Y.,and “Current Protocols in Molecular Biology”, Ausubel et al., John Wiley& Sons, 1989, New York, N.Y.

[0099] A variety of expression vector/host systems may be utilized tocontain and express ASIC2A and/or ASIC3 coding sequences. These include,but are not limited to, microorganisms such as bacteria transformed withrecombinant bacteriophage, plasmid, or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with virus expression vectors (e.g., baculovirus); plant cellsystems transformed with virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterialexpression vectors (e.g., Ti or pBR322 plasmids); or animal cellsystems.

[0100] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the Bluescript® phagemid (Stratagene, LaJolla, Calif.) or pSport1™ plasmid (Gibco BRL) and ptrp-lac hybrids, andthe like may be used. Other preferred prokaryotic vectors include butare not limited to pQE-9, pQE60, pQE70 (Quiagen), pNH8A, pNH16a, pNH18a,pNH46A (Stratagene) ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). The baculovirus polyhedrin promoter may be used in insectcells. Promoters or enhancers derived from the genomes of plant cells(e.g., heat shock, RUBISCO; and storage protein genes) or from plantviruses (e.g., viral promoters or leader sequences) may be cloned intothe vector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding ASIC2Aand/or ASIC3, vectors based on SV40 or EBV may be used with anappropriate selectable marker.

[0101] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for ASIC2A and/or ASIC3. Forexample, when large quantities of ASIC2A and/or ASIC3 are needed for theinduction of antibodies, vectors, which direct high level expression offusion proteins that are readily purified, may be used. Such vectorsinclude, but are not limited to, the multifunctional E. coli cloning andexpression vectors such as Bluescript® (Stratagene, La Jolla, Calif.),into which the sequence encoding ASIC2A or ASIC3 may be ligated in framewith sequences for the amino-terminal Methionine and the subsequent 7residues of β-galactosidase so that a hybrid protein is produced; pINvectors (Van Heeke and Schuster, J. Biol. Chem. 1989; 264: 5503); andthe like; pGEX vectors (Promega, Madison, Wis.) may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems may be designed to includeheparin, thrombin, or factor XA protease cleavage sites so that thecloned polypeptide of interest can be released from the GST moiety atwill.

[0102] In addition to bacteria, eucaryotic microbes, such as yeast, mayalso be used as hosts. Laboratory strains of Saccharomyces cerevisiae,Baker's yeast, are most used although a number of other strains orspecies are commonly available. Vectors employing, for example, the 2μorigin of replication of Broach et al. (Meth Enzymol 1983; 101: 307), orother yeast compatible origins of replication (see, for example,Stinchcomb et al. Nature 1979: 282; 39, Tschumper et al., Gene 1980: 10;157, Clarke et al., Meth Enzymol 1983; 101: 300) may be used. Controlsequences for yeast vectors include promoters for the synthesis ofglycolytic enzymes (Hess et al. J Adv Enzyme Reg 1968; 7: 149; Hollandet al., Biochemistry 1978; 17: 4900). Additional promoters known in theart include the promoter for 3-phosphoglycerate kinase (Hitzeman et al.,J Biol Chem 1980; 255: 2073), alcohol oxidase, and PGH. Other promoters,which have the additional advantage of transcription controlled bygrowth conditions and/or genetic background are the promoter regions foralcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradativeenzymes associated with nitrogen metabolism, the alpha-factor system andenzymes responsible for maltose and galactose utilization. It is alsobelieved terminator sequences are desirable at the 3′ end of the codingsequences. Such terminators are found in the 3′ untranslated regionfollowing the coding sequences in yeast-derived genes. For reviews, see“Current Protocols in Molecular Biology”, Ausubel et al., John Wiley &Sons, 1989, New York, N.Y. and Grant et al., Meth Enzymol. 1987; 153:516.

[0103] In cases where plant expression vectors are used, the expressionof a sequence encoding ASIC2A or ASIC3 may be driven by any of a numberof promoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu et al., EMBO J. 1987; 6: 307;Brisson et al., Nature 1984; 310: 511). Alternatively, plant promoterssuch as the small subunit of RUBISCO or heat shock promoters may be used(Coruzzi et al., EMBO J. 1984; 3: 1671; Broglie et al., Science 1984;224: 838; Winter et al., Results Probl. Cell Differ 1991; 17: 85). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs S or Murry L E in “McGraw Hill Yearbook of Science and Technology”McGraw Hill, 1992, New York, N.Y.; pp. 191-196 or Weissbach andWeissbach in “Methods for Plant Molecular Biology”, Academic Press,1988, New York, N.Y.; pp. 421-463).

[0104] An insect system may also be used to express ASIC2A and/or ASIC3.For example, in one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The sequenceencoding ASIC2A and/or ASIC3 may be cloned into a non-essential regionof the virus, such as the polyhedrin gene, and placed under control ofthe polyhedrin promoter. Successful insertion of ASIC2A or ASIC3 willrender the polyhedrin gene inactive and produce recombinant viruslacking coat protein. The recombinant viruses may then be used toinfect, for example, S. frugiperda cells or Trichoplusia larvae in whichASIC2A and/or ASIC3 may be expressed (Smith et al., J Virol 1983; 46:584; Engelhard et al., Proc Natl Acad Sci 1994; 91: 3224).

[0105] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, a sequence encoding ASIC2A and/or ASIC3 may beligated into an adenovirus transcription/translation complex consistingof the late promoter and tripartite leader sequence. Insertion in anon-essential E1 or E3 region of the viral genome may be used to obtaina viable virus, which is capable of expressing ASIC2A and/or ASIC3 ininfected host cells (Logan and Shenk, Proc Natl Acad Sci 1984; 81:3655). In addition, transcription enhancers, such as the Rous sarcomavirus (RSV) enhancer, may be used to increase expression in mammalianhost cells.

[0106] Specific initiation signals may also be used to achieve moreefficient translation of a sequence encoding ASIC2A and/or ASIC3. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding ASIC2A or ASIC3, together with theirinitiation codon, and upstream sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a portion thereof, is inserted, exogenoustranslational control signals including the ATG initiation codon shouldbe provided. Furthermore, the initiation codon should be in the correctreading frame to ensure the correct translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf et al., Results Probl Cell Differ 1994; 20: 125;Bittner et al., Meth Enzymol 1987; 153: 516).

[0107] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, WI38,and COS, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

[0108] In a preferred expression system, cDNAs or cRNAs encoding ASIC2Aand/or ASIC3 are coinjected directly into Xenopus oocytes, cDNAs intonuclei and cRNA into the cytoplasm, thereby allowing for in vitrotranslation and assembly into a functional heteromultimeric proton-gatedchannel capable of demonstrating functional characteristics of nativeproton-gated channels including ion selectivity, gating-kinetics, ligandpreferences, and sensitivity to pharmacological agents such as amiloridefor a model assay which mimics in vivo characteristics.

[0109] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines, which stablyexpress ASIC2A or ASIC3, or both ASIC2A and ASIC3, may be transformedusing expression vectors which may contain viral origins of replicationand/or endogenous expression elements and a selectable marker gene onthe same or separate vector. Following the introduction of the vector,cells may be allowed to grow for 1-2 days in an enriched media beforethey are switched to selective media. The purpose of the selectablemarker is to confer resistance to selection, and its presence allowsgrowth and recovery of cells, which successfully express the introducedsequences. Resistant clones of stably transformed cells may beproliferated using tissue culture techniques appropriate to the celltype.

[0110] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler et al., Cell 1977; 11:223) and adenine phospho-ribosyltransferase (Lowy et al., Cell 1980; 22:817) genes which can be employed in tk+- or aprt+-cells, respectively.Also, antimetabolite, antibiotic, or herbicide resistance can be used asthe basis for selection; for example, dhfr, which confers resistance tomethotrexate (Wigler et al., Proc Natl Acad Sci 1980; 77: 3567); npt,which confers resistance to the aminoglycosides neomycin and G-418(Colbere-Garapin et al., J Mol Biol 1981; 150: 1) and als or pat, whichconfer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry L E, supra). Additionalselectable genes have been described, for example, trpB, which allowscells to utilize indole in place of tryptophan, or hisD, which allowscells to utilize histinol in place of histidine (Hartman and Mulligan,Proc Natl Acad Sci 1988; 85: 8047). Recently, the use of visible markershas gained popularity with such markers as anthocyanins, β-glucuronidaseand its substrate GUS, and luciferase and its substrate luciferin, beingwidely used not only to identify transformants, but also to quantify theamount of transient or stable protein expression attributable to aspecific vector system (Rhodes et al., Methods Mol Biol 1995; 55: 121).

[0111] Although the presence/absence of marker for gene expressionsuggests that the gene of interest is also present, its presence andexpression may need to be confirmed. For example, if the sequenceencoding ASIC2A and/or ASIC3 is inserted within a marker gene sequence,recombinant cells containing sequences encoding ASIC2A and/or ASIC3 canbe identified by the absence of marker gene function. Alternatively, amarker gene can be placed in tandem with a sequence encoding ASIC2A orASIC3 under the control of a single promoter. Expression of the markergene in response to induction or selection usually indicates expressionof the tandem gene as well.

[0112] Alternatively, host cells, which contain the coding sequences forASIC2A and/or ASIC3 and express both ASIC2A and ASIC3 may be identifiedby a variety of procedures known to those of skill in the art. Theseprocedures include, but are not limited to, DNA-DNA or DNA-RNAhybridizations and protein bioassay or immunoassay techniques, whichinclude membrane, solution, or chip based technologies for the detectionand/or quantification of the nucleic acid or protein.

[0113] The presence of the polynucleotide sequences encoding ASIC2Aand/or ASIC3 can be detected by DNA-DNA or DNA-RNA hybridization oramplification using probes or portions or fragments of polynucleotidesencoding ASIC2A or ASIC3. Nucleic acid amplification based assaysinvolve the use of oligonucleotides or oligomers based on the ASIC2A- orASIC3-encoding sequences to detect transformants containing DNA or RNAencoding ASIC2A and/or ASIC3. As used herein “oligonucleotides” or“oligomers” refer to a nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides, preferably about 15 to30 nucleotides, and more preferably about 20-25 nucleotides, which canbe used as a probe or amplimer.

[0114] A variety of protocols for detecting and measuring theco-expression of ASIC2A and ASIC3, using either polyclonal or monoclonalantibodies specific for each protein are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescent activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on ASIC2A or ASIC3 is preferred, but acompetitive binding assay may be employed. These and other assays aredescribed, among other places, in “Serological Methods: A LaboratoryManual”, Hampton et al., APS Press, 1990, St-Paul, Mich. and Maddox etal., J Exp Med 1983; 158: 1211).

[0115] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encodingASIC2A or ASIC3 include oligo-labeling, nick translation, end-labelingor PCR amplification using a labeled nucleotide. Alternatively, thesequence encoding ASIC2A or ASIC3, or any portion thereof, may be clonedinto a vector for the production of an mRNA probe. Such vectors areknown in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3 or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits: from e.g. Pharmacia & Upjohn, (Kalamazoo, Mich.); Promega (MadisonWis.); and U.S. Biochemical Corp. (Cleveland, Ohio), or Ambion (Austin,Tex.). Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

[0116] Host cells co-transformed with nucleotide sequences encoding bothASIC2A and/or ASIC3 may be cultured under conditions suitable for theexpression and recovery of the proteins from cell culture. The proteinsproduced by a recombinant cell may be secreted or containedintracellularly depending on the sequence and/or the vector used. Aswill be understood by those of skill in the art, expression vectorscontaining polynucleotides, which encode ASIC2A or ASIC3 may be designedto contain signal sequences which direct secretion of ASIC2A and/orASIC3 through a prokaryotic or eukaryotic cell membrane.

[0117] Other recombinant constructions may be used to join sequencesencoding ASIC2A or ASIC3 to nucleotide sequence encoding a polypeptidedomain, which will facilitate purification of the expressed proteins.Such purification facilitating domains include, but are not limited to,metal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp.,Seattle, Wash.). The inclusion of cleavable linker sequences such asthose specific for Factor XA or enterokinase (Invitrogen, San Diego,Calif.) between the purification domain and ASIC2A or ASIC3 may be usedto facilitate purification. One such expression vector provides forexpression of a fusion protein containing ASIC2A and/or ASIC3, athioredoxin or an enterokinase cleavage site, and followed by sixhistidine residues. The histidine residues facilitate purification onIMIAC (immobilized metal ion affinity chromatography as described inPorath et al., Prot Exp Purif 1992; 3: 263) while the enterokinasecleavage site provides a means for purifying ASIC2A or ASIC3 from thefusion protein. A discussion of vectors which contain fusion proteins isprovided in Kroll et al. (DNA Cell Biol 1993; 12:441).

[0118] In addition to recombinant production, fragments of ASIC2A and/orASIC3 may be produced by direct peptide synthesis using solid-phasetechniques (see Stewart et al., “Solid-Phase Peptide Synthesis”, W HFreeman & Co., 1969, San Francisco, Calif.; Merrifield et al., J Am ChemSoc 1963; 85: 2149). Chemical synthesis may be performed using manualtechniques or by automation. Automated synthesis may be achieved, forexample, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Various fragments of ASIC2A and/or ASIC3 may be chemicallysynthesized separately and combined using chemical methods to producethe full-length molecule.

[0119] Thus as set forth herein the invention includes the provision ofa novel subfamily of heteromultimeric proton-gated ion channels asexemplified by the novel association of ASIC2A and ASIC3 polypeptides,encoded, respectively, by nucleic acids of SEQ ID NO: 1 or NO:5 and SEQID NO: 3 or NO:7 as well as DNA sequences which hybridize thereto understringent hybridization conditions, and DNA sequences encoding the sameallelic variant or analogue proton-gated channel protein through use ofat least in part degenerate codons. The ASIC-2S.2 channel complex canalso be used to located and identify other closely related members ofthis subfamily as described in Cannessa et al (Nature 1994; 367: 463).

[0120] Polypeptides of SEQ ID NO:2, NO:4, NO:6 and NO:8, as well as anyprotein, protein fragment, synthetic protein or peptide thereof areprojected to have uses earlier described including therapeutic,diagnostic, and prognostic assays and protocols and will provide thebasis for monoclonal and polyclonal antibodies specifically reactivewith the channel protein.

[0121] Therapeutics

[0122] In another embodiment of the invention, ASIC-2S.2heteromultimeric channels may be used for therapeutic purposes. Based onthe mRNA distribution patterns of ASIC2A and ASIC3 showing that ASIC2Aand ASIC3 transcripts are primarily but not exclusively associated withcells of the peripheral and central nervous systems, ASIC-2S.2 isbelieved to play a role in the regulation of neurotransmitter release,neuronal excitability, or excitotoxicity. Indeed, secretory granules andsynaptic vesicules are known to contain high concentrations of protons(low intravesicular pH), which are co-released with otherneurotransmitters during regulated and constitutive exocytosis. Releasedprotons might thus activate pre- and/or post-synaptic, or extrasynapticASIC-2S.2 receptors. Indeed, under certain conditions, low pH orextracellular acidosis has been shown to influence synaptic transmissionas well as the induction of long-term potentiation (Igelmund et al.,Brain Res 1995; 689: 9; Velisek et al., Hippocampus 1998; 8: 24). Also,in certain animal seizure models, neuroprotective effects of low pH havebeen observed (Velisek et al., Exp Brain Res 1994; 101: 44). Thus, animportant use of ASIC-2S.2 is screening for compounds that regulateneurotransmitter release, synaptic efficacy, neuroexcitability, orneurotoxicity. Such compounds may have utility in a number ofphysiological and pathological situations pertaining, for example, tocognition, perception, learning, memory, pain and many others. Morespecifically, in situ hybridization and duplex RT-PCR analysis (FIGS. 4and 5) indicate that coexpression of ASIC2A and ASIC3 is regionspecific. Interestingly, in contrast to what is reported for the rat, wereport herein for the first time that ASIC2A is highly expressed inhuman sensory ganglia, such as the trigeminal ganglia. Furthermore, thehighest probability of coexpression of ASIC2A and ASIC3 has also beenfound in these sensory ganglia. This strongly suggests an important rolefor the ASIC-2S.2 channels in pain and/or somato-sensory transmission.

[0123] In one embodiment, antagonists or inhibitors of the ASIC-2S.2protein complex or vectors expressing antisense sequences may be used totreat disorders and diseases of the nervous system resulting fromaltered ion transport, signal transmission, and apoptosis. Such diseasesinclude, but are not limited to, chronic pain, neuropathic pain such asdiabetic-, cancer-, and AIDS-related, neurodegenerative diseases such asAlzheimer's disease, Parkinson's disease, Huntington's disease,Creutzfeld-Jacob disease, and amyotrophic lateral sclerosis, anddementias, including AIDS-related, as well as convulsions, epilepsy,stroke, and anxiety and depression.

[0124] In another embodiment, antagonists or inhibitors of the ASIC-2S.2protein complex or vectors expressing antisense sequences may be used totreat cardiovascular diseases such as angina, congestive heart failure,vasoconstriction, hypertension, atherosclerosis, restenosis, andbleeding.

[0125] Agonists, which enhance the activity and antagonists, which blockor modulate the effect of ASIC-2S.2 may be used in those situationswhere such enhancement or inhibition is therapeutically desirable. Suchagonists, antagonists or inhibitors may be produced using methods, whichare generally known in the art, such as screening libraries ofpharmaceutical agents for compounds, which directly (or indirectly) andspecifically interact and/or bind ASIC-2S.2. Other methods involve theuse of purified ASIC2A and/or ASIC3 to produce antibodies. For example,in one aspect, antibodies which are specific for ASIC2A or ASIC3, orASIC-2S.2 may be used directly as an antagonist of ASIC-2S.2, orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue, which express ASIC-2S.2.

[0126] The antibodies may be generated using methods that are well knownin the art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0127] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith ASIC2A, or ASIC3, or with ASIC2A and ASIC3, including covalentlylinked ASIC2A-ASIC3 tandems and ASIC2A-ASIC3 chimers, or any fragment oroligopeptide thereof which has immunogenic properties. Depending on thehost species, various adjuvants may be used to increase immunologicalresponse. Such adjuvants include, but are not limited to, Freund's,mineral gels such as aluminum hydroxide, and surface active substancessuch as lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, keyhole limpet hemocyanin, and dinitrophenol. Among adjuvantsused in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvumare especially preferable.

[0128] It is preferred that the peptides, fragments, or oligopeptidesused to induce antibodies to ASIC-2S.2 have an amino acid sequenceconsisting of at least five amino acids, and more preferably at least 10amino acids. It is also preferable that they are identical to a portionof the amino acid sequence of the natural protein, and they may containthe entire amino acid sequence of a small, naturally occurring molecule.Short stretches of ASIC2A or ASIC3 amino acids may be fused with thoseof another protein such as keyhole limpet hemocyanin and antibodyproduced against the chimeric molecule.

[0129] Monoclonal antibodies to ASIC-2S.2 may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Koehler et al. Nature 1975; 256: 495; Kosbor etal., Immunol Today 1983; 4: 72; Cote et al., Proc Natl Acad Sci 1983;80: 2026; Cole et al., “Monoclonal Antibodies and Cancer Therapy”, AlanR. Liss Inc., 1985, New York, N.Y., pp. 77-96).

[0130] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison et al. (1984) Proc. Natl.Acad. Sci. 81:6851-6855; Neuberger et al. (1984) Nature 312:604-608;Takeda et al. (1985) Nature 314:452-454). Alternatively, techniquesdescribed for the production of single chain antibodies may be adapted,using methods known in the art, to produce ASIC-2S.2-specific singlechain antibodies. Antibodies with related specificity, but of distinctidiotypic composition, may be generated by chain shuffling from randomcombinatorial immunoglobin libraries (Burton, D. R. (1991) Proc. Natl.Acad. Sci. 88:11120-3).

[0131] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

[0132] Antibody fragments which contain specific binding sites forASIC-2S.2 may also be generated. For example, such fragments include,but are not limited to, the F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse et al. (1989) Science 256:1275-1281).

[0133] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between ASIC-2S.2 and its specific antibody. Atwo-site, monoclonal-based immunoassay utilizing monoclonal antibodiesreactive to two non-interfering ASIC-2S.2 epitopes is preferred, but acompetitive binding assay may also be employed (Maddox, supra).

[0134] In another embodiment of the invention, the polynucleotidesencoding ASIC2A and/or ASIC3, or any fragment thereof, or antisensesequences, may be used for therapeutic purposes. In one aspect,antisense to the polynucleotide encoding ASIC2A and/or ASIC3 may be usedin situations in which it would be desirable to block the synthesis ofthe ASIC-2S.2 protein complex. In particular, cells may be transformedwith sequences complementary to polynucleotides encoding ASIC2A and/orASIC3. Thus, antisense sequences may be used to modulate ASIC-2S.2activity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligomers or largerfragments, can be designed from various locations along the coding orcontrol regions of sequences encoding ASIC2A and/or ASIC3.

[0135] Expression vectors derived from retroviruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods, which are well known to those skilled in the art,can be used to construct recombinant vectors which will expressantisense polynucleotides of the genes encoding ASIC2A and/or ASIC3.These techniques are described both in Sambrook et al. (supra) and inAusubel et al. (supra).

[0136] Genes encoding ASIC2A and/or ASIC3 can be turned off bytransforming a cell or tissue with expression vectors which express highlevels of a polynucleotide or fragment thereof which encodes ASIC2Aand/or ASIC3. Such constructs may be used to introduce untranslatablesense or antisense sequences into a cell. Even in the absence ofintegration into the DNA, such vectors may continue to transcribe RNAmolecules until all copies are disabled by endogenous nucleases.

[0137] Transient expression may last for a month or more with anon-replicating vector and even longer if appropriate replicationelements are part of the vector system.

[0138] As mentioned above, modifications of gene expression can beobtained by designing antisense molecules, DNA, RNA or PNA, to thecontrol regions of the gene encoding ASIC2A or ASIC3, i.e., thepromoters, enhancers, and introns. Oligonucleotides derived from thetranscription initiation site, e.g., between positions −10 and +10 fromthe 5′ end of the transcript, are preferred. Similarly, inhibition canbe achieved using “triple helix” base-pairing methodology. Triple helixpairing is useful because it causes inhibition of the ability of thedouble helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA have been described in the literature (Gee,J. E. et al. (1994) In: Huber, B. E. and Carr, B. I. Molecular andImmunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.). Theantisense molecules may also be designed to block translation of mRNA bypreventing the transcript from binding to ribosomes.

[0139] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding ASIC2A and/or ASIC3.

[0140] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0141] Antisense molecules and ribozymes of the invention may beprepared by any method known in the art for the synthesis of RNAmolecules. These include techniques for chemically synthesizingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences encoding ASIC2A and/or ASIC3. Such DNAsequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize antisense RNA constitutively orinducibly can be introduced into cell lines, cells, or tissues.

[0142] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0143] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection and by liposomeinjections may be achieved using methods which are well known in theart.

[0144] Any of the therapeutic methods described above may be applied toany suitable subject including, for example, mammals such as dogs, cats,cows, horses, rabbits, monkeys, and most preferably, humans.

[0145] An additional embodiment of the invention relates to theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist ofASIC-2S.2, or any component thereof, antibodies to ASIC-2S.2, mimetics,agonists, antagonists, or inhibitors of ASIC-2S.2. The compositions maybe administered alone or in combination with at least one other agent,such as stabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water. The compositions may beadministered to a patient alone, or in combination with other agents,drugs or hormones.

[0146] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0147] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0148] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0149] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0150] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0151] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0152] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hank's solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances, which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0153] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0154] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0155] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0156] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of ASIC-2S.2, or any componentthereof, such labeling would include amount, frequency, and method ofadministration.

[0157] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0158] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rats, rabbits, dogs, or pigs.The animal model may also be used to determine the appropriateconcentration range and route of administration. Such information canthen be used to determine useful doses and routes for administration inhumans.

[0159] A therapeutically effective dose refers to that amount of activeingredient, for example ASIC-2S.2 or any component or fragment thereof,antibodies against ASIC-2S.2, agonists, antagonists or inhibitors ofASIC-2S.2, which ameliorates the symptoms or condition. Therapeuticefficacy and toxicity may be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED₅₀ (thedose therapeutically effective in 50% of the population) and LD₅₀ (thedose lethal to 50% of the population). The dose ratio betweentherapeutic and toxic effects is the therapeutic index, and it can beexpressed as the ratio, LD₅₀/ED₅₀.

[0160] Pharmaceutical compositions, which exhibit large therapeuticindices are preferred. The data obtained from cell culture assays andanimal studies is used in formulating a range of dosage for human use.The dosage contained in such compositions is preferably within a rangeof circulating concentrations that include the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0161] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0162] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0163] Diagnostics

[0164] In another embodiment, antibodies which specifically bindASIC-2S.2 may be used for the diagnosis of conditions or diseasescharacterized by expression of ASIC-2S.2, or in assays to monitorpatients being treated with ASIC-2S.2 agonists, antagonists orinhibitors. The antibodies useful for diagnostic purposes may beprepared in the same manner as those described above for therapeutics.Diagnostic assays for ASIC-2S.2 include methods which utilize theantibody and a label to detect ASIC-2S.2, or any component therof, inhuman body fluids or extracts of cells or tissues. The antibodies may beused with or without modification, and may be labeled by joining them,either covalently or non-covalently, with a reporter molecule. A widevariety of reporter molecules which are known in the art may be used,several of which are described above.

[0165] A variety of protocols including ELISA, RIA, and FACS formeasuring ASIC-2S.2 are known in the art and provide a basis fordiagnosing altered or abnormal levels of ASIC-2S.2 expression. Normal orstandard values for ASIC-2S.2 expression are established by combiningbody fluids or cell extracts taken from normal mammalian subjects,preferably human, with antibody to ASIC-2S.2 under conditions suitablefor complex formation. The amount of standard complex formation may bequantified by various methods, but preferably by photometric, means.Quantities of ASIC-2S.2 expressed in subject, control and disease,samples from biopsied tissues are compared with the standard values.Deviation between standard and subject values establishes the parametersfor diagnosing disease.

[0166] In another embodiment of the invention, the polynucleotidesencoding ASIC-2S.2 may be used for diagnostic purposes. Thepolynucleotides which may be used include oligonucleotide sequences,antisense RNA and DNA molecules, and PNAs. The polynucleotides may beused to detect and quantitate gene expression in biopsied tissues inwhich expression of ASIC-2S.2 may be correlated with disease. Thediagnostic assay may be used to distinguish between absence, presence,and excess expression of ASIC-2S.2, and to monitor regulation ofASIC-2S.2 levels during therapeutic intervention. The diagnostic assaymay also be used to determine the ratio of expression between ASIC2A andASIC3, and any changes in these expression rations, as an index ormarker of ASIC-2S.2 expression.

[0167] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences encodingASIC2A or ASIC3 or closely related molecules, may be used to identifynucleic acid sequences which encode ASIC2A or ASIC3. The specificity ofthe probe, whether it is made from a highly specific region, e.g., 10unique nucleotides in the 5′ regulatory region, or a less specificregion, e.g., especially in the 3′ coding region, and the stringency ofthe hybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding ASIC2A or ASIC3, alleles, or related sequences.

[0168] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the ASIC2A or ASIC3 encoding sequences. The hybridization probes ofthe subject invention may be DNA or RNA and derived from the nucleotidesequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO:7, orfrom genomic sequences including promoter, enhancer elements, andintrons of the naturally occurring ASIC2A and ASIC3.

[0169] Means for producing specific hybridization probes for DNAsencoding ASIC2A or ASIC3 include the cloning of nucleic acid sequencesencoding ASIC2A or ASIC3 derivatives into vectors for the production ofmRNA probes. Such vectors are known in the art, commercially available,and may be used to synthesize RNA probes in vitro by means of theaddition of the appropriate RNA polymerases and the appropriate labelednucleotides. Hybridization probes may be labeled by a variety ofreporter groups, for example, radionuclides such as ³²P or ³⁵S, orenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems, and the like.

[0170] Polynucleotide sequences encoding ASIC2A and/or ASIC3 may be usedfor the diagnosis of conditions or diseases which are associated withexpression of ASIC-2S.2. Examples of such conditions or diseases includeneurological diseases including chronic pain, neuropathic pain such asdiabetic-, cancer-, and AIDS-related neurodegenerative diseases such asAlzheimer's disease, Parkinson's disease, Huntington's disease,Creutzfeld-Jacob disease, and amyotrophic lateral sclerosis, anddementias, such as AIDS-related, as well as convulsions, epilepsy,stroke, and anxiety and depression, cardiovascular diseases such asangina, congestive heart failure, vasoconstriction, hypertension,atherosclerosis, restenosis, and bleeding. The polynucleotide sequencesencoding ASIC2A or ASIC3 may be used in Southern or northern analysis,dot blot, or other membrane-based technologies; in PCR technologies; orin dip stick, pin, ELISA or chip assays utilizing fluids or tissues frompatient biopsies to detect altered ASIC-2S.2 expression. Suchqualitative or quantitative methods are well known in the art.

[0171] In a particular aspect, the nucleotide sequences encoding ASIC2Aand/or ASIC3 may be useful in assays as probes that detect activation orinduction of various neurological or other non-neurological disorders,particularly those mentioned above. The nucleotide sequence encodingASIC2A and/or ASIC3 may be labelled by standard methods and added to afluid or tissue sample from a patient under conditions suitable for theformation of hybridization complexes. After a suitable incubationperiod, the sample is washed and the signal is quantified and comparedwith a standard value. If the amount of signal in the biopsied orextracted sample is significantly different from that of a comparablecontrol sample, this indicates that the levels of nucleotide sequencesthat hybridized with the labelled probe in the sample are alsodifferent. The presence of altered levels of nucleotide sequencesencoding ASIC2A and/or ASIC3 in the sample indicates the presence of theassociated disease. Such assays may also be used to evaluate theefficacy of a particular therapeutic treatment regimen in animalstudies, in clinical trials, or in monitoring the treatment of anindividual patient.

[0172] In order to provide a basis for the diagnosis of diseaseassociated with expression of ASIC-2S.2, a normal or standard profilefor expression is established. This may be accomplished by combiningbody fluids or cell extracts taken from normal subjects, either animalor human, with a sequence, or a fragment thereof, which encodes ASIC2Aand/or ASIC3 under conditions suitable for hybridization oramplification. Standard hybridization may be quantified by comparing thevalues obtained from normal subjects with those from an experiment wherea known amount of a substantially purified polynucleotide is used.Standard values obtained from normal samples may be compared with valuesobtained from samples from patients who are symptomatic for disease.Deviation between standard and subject values is used to establish thepresence of disease.

[0173] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0174] With respect to neurological diseases, the presence of arelatively high amount of transcript in biopsied tissue from anindividual may indicate a predisposition for the development of thedisease, or may provide a means for detecting the disease prior to theappearance of actual clinical symptoms. A more definitive diagnosis ofthis type may allow health professionals to employ preventive measuresor aggressive treatment earlier thereby preventing the development orfurther progression of the disease.

[0175] Additional diagnostic uses for oligonucleotides encoding ASIC2Aand/or ASIC3 may involve the use of PCR. Such oligomers may bechemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation and another with antisense,employed under optimised conditions for identification of a specificgene or condition. The same two oligomers, nested sets of oligomers, oreven a degenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantification of closely related DNA orRNA sequences.

[0176] Methods which may also be used to quantify the expression ofASIC-2S.2 include radiolabelling or biotinylating nucleotides,coamplification of a control nucleic acid, and standard curves ontowhich the experimental results are interpolated (Melby P C et al. JImmunol Methods, 1993; 159: 235; Duplaa C et al. Anal Biochem 1993;229). The speed of quantification of multiple samples may be acceleratedby running the assay in an ELISA format where the oligomer of interestis presented in various dilutions and a spectrophotometric orcalorimetric response gives rapid quantification.

[0177] Screening Assays

[0178] In another embodiment of the invention, ASIC-2S.2, its active,catalytic or immunogenic fragments or oligopeptides thereof, can be usedfor screening libraries of compounds in any of a variety of drugscreening techniques. The fragment employed in such screening may befree in solution, affixed to a solid support, borne on a cell surface,or located intracellularly. The formation of binding complexes, betweenASIC-2S.2 and the agent being tested, may be measured. Thus, thepolypeptides derived from ASIC-2S.2, or any component thereof, may alsobe used to assess the binding of small molecule substrates and ligandsin, for example, cells, cell-free preparations, chemical libraries, andnatural product mixtures. These substrates and ligands may be naturalsubstrates and ligands or may be structural or functional mimetics. Ingeneral, such screening procedures involve producing appropriate cells,which express the receptor polypeptide complex of the present inventionon the surface thereof. Such cells include cells from mammals, yeast,insects (e.g. Drosophila) or bacteria (e.g. E. coli). Cells expressingthe receptor (or cell membranes containing the expressed receptor) arethen contacted with a test compound to observe binding, or stimulationor inhibition of a functional response (for example inhibition ofproton-activated currents).

[0179] The assays may simply test binding of a candidate compoundwherein adherence to the cells bearing the receptor is detected by meansof a label directly or indirectly associated with the candidate compoundor in an assay involving competition with a labelled competitor.Further, these assays may test whether the candidate compound results ina signal generated by activation of the receptor, using detectionsystems appropriate to the cells bearing the receptor at their surfaces(for example increased ion permeation measured by patch clamp or,preferably by ion imaging with ion-specific dyes). Inhibitors ofactivation are generally assayed in the presence of a known agonist (forexample protons) and the effect of the candidate compound on theactivation by the agonist is observed. Standard methods for conductingsuch screening assays are well understood in the art. Typically, theresponse may be measured by use of a microelectrode techniqueaccompanied by such measurement strategies as voltage clamping of thecell whereby activation of ion channels may be identified by inward oroutward current flow as detected using the microelectrodes. ²²Na, ⁸⁶Rb,⁴⁵Ca radiolabelled cations or ¹⁴C or ³H guanidine may be used to assesssuch ion flux; a sodium, calcium or potassium ion sensitive dye (such asFura-2, or Indo) may also be used to monitor ion passage through thereceptor ion channel, or a potential sensitive dye may be used tomonitor potential changes, such as in depolarisation.

[0180] Alternatively, it is also possible to mutate the ASIC2A and/orASIC3 cDNA in order to produce a constituvely active ASIC-2S.2 channel,as has been shown with other DEG/ENaC family members (Huang et al.,Nature 367: 467; Waldman et al., J Biol Chem 1997: 271; 10433, Sakai etal., J Physiol 1999; 519: 323, Schaefer et al., FEBS Lett 2000; InPress). Then, the constitutively active channel may be expressed in hostcells to produce a screening assay where channel activity is permanent.The recording of channel activity my be carried out either by membranevoltage analysis, directly (patch clamp, for example) or indirectly(fluorescent probes, for example) or by sodium entry measurement(radioactive sodium influx, fluorescent probes, or reporter genes).

[0181] Another technique for drug screening, which may be used, providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication WO84/03564. In this method, as applied to ASIC-2S.2, largenumbers of different small test compounds are synthesised on a solidsubstrate, such as plastic pins or some other surface. The testcompounds are reacted with ASIC-2S.2, or components, or fragmentsthereof, and washed. Bound ASIC-2S.2 is then detected by methods wellknown in the art. Purified ASIC-2S.2, or any component thereof, can alsobe coated directly onto plates for use in the aforementioned drugscreening techniques. Alternatively, non-neutralising antibodies can beused to capture the peptide and immobilise it on a solid support.

[0182] In another embodiment, one may use competitive drug screeningassays in which neutralising antibodies capable of binding ASIC-2S.2specifically compete with a test compound for binding ASIC-2S.2. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with ASIC-2S.2.

[0183] In additional embodiments, the nucleotide sequences which encodethe individual components of ASIC-2S.2, namely ASIC2A and ASIC3, may beused in any molecular biology or pharmacology techniques that have yetto be developed, provided the new techniques rely on properties of thenucleotide or polypeptide sequences that are currently known, including,but not limited to, such properties as the triplet genetic code andspecific base pair interactions.

EXAMPLES

[0184] The following examples are intended to further illustrate theinvention and are not intended to limit the scope of the invention inany way. All references cited herein, whether previously or in thefollowing examples, are expressly incorporated in their entirety byreference. All oligonucleotides disclosed in the following examples aredesigned using two recognised software packages: GeneWorks 2.5.1 andMacVector 6.0.1 (Oxford Molecular).

Example 1 Vector Constructs for the Functional Expression of ASIC-2S.2Channels

[0185] Method 1: Expression of ASIC-2S.2 is accomplished by introducinginto appropriate host cells, by various injection or transfectiontechniques known to one skilled in the art, two separate vectorscomprising the nucleic acids encoding, respectively, the ASIC2A andASIC3 polypeptides of SEQ ID NO:2 or NO:6 and SEQ ID NO: 4 or NO:8. Apreferred eukaryotic expression vector is pcDNA3 (InVitrogen), or anyderivatives thereof. Based on nucleic acid sequences of SEQ ID NO:1, SEQID NO:3, SEQ ID NO:5 and SEQ ID NO:7, specific oligonucleotide primersare designed immediately upstream and downstream, respectively, of theinitiation and the stop codons. All primers are extended to addartificial restriction sites (e.g. forward primers with EcoRI andreverse primers with XbaI), allowing RT-PCR amplified full lengthnucleic acids to be directionally subcloned into pcDNA3. Ligatedproducts are used to transform E. Coli strain DH5α from which purifiedplasmids are prepared using commercially available kits (Quiagen).

[0186] Method 2: Expression of ASIC-2S.2 is achieved by introducing intoappropriate host cells, by various injection or transfection techniquesknown to one skilled in the art, a biscistronic vector comprising twonucleic acids encoding, respectively, ASIC2A and ASIC3 polypeptides ofSEQ ID NO:2 or NO:6 and SEQ ID NO: 4 or NO:8. Bicistronic expressionvectors produce one transcript with two translation initiation points,resulting in the simultaneous expression of two genes of interest. Apreferred bicistronic vector is pIRES (Clontech) which permits thesubcloning of two distinct genes in two separate multiple cloning sites(MCS A and B) located on either side of the internal ribosome entry site(IRES) from the encephalomyocarditis virus (ECMV). This allows thetranslation of two consecutive open reading frames from the samemessenger RNA (Jang S K et al. J. Virol. 1990; 62: 2636—Rees S et al.:BioTechniques 1996; 20: 102). The MCSs and IRES sequences are downstreamof the immediate early promoter of cytomegalovirus (PCMV IE). Theintervening sequence (IVS) between PCMV IE and the MCS is an intron thatis efficiently spliced out following transcription. SV40 polyadenylationsignals downstream of the MCS direct proper processing of the 3′ end ofthe mRNA from subcloned genes. Bacteriophage T7 and T3 promoters arelocated upstream and downstream of MCS A and B, respectively. pIRES usesthe neomycin resistance gene (Neor) to permit selection of transformedcells. Neor is expressed from the SV40 enhancer/promoter, and asynthetic polyadenylation signal directs proper processing of the 3′ endof the Neor mRNA. The SV40 origin also allows for replication inmammalian cells expressing the SV40 T antigen. The vector backbone alsocontains the β-lactamase gene for ampicillin resistance and a ColE1origin of replication for propagation in E. coli and a f1 origin forsingle-stranded DNA production. As described above in method 1, RT-PCRamplified ASIC2A and ASIC3 nucleic acids are directionally subcloned,respectively, into MCS A and MCS B (or vice versa). Control vectorscomprising two copies of ASIC2A- or ASIC3-encoding nucleic acids arealso constructed. Ligated products are used to transform E. Coli strainDH5α from which purified plasmids are prepared using commerciallyavailable kits (Quiagen).

[0187] Method 3: Expression of ASIC-2S.2 is achieved by introducing intoappropriate host cells, by various injection or transfection techniquesknown to one skilled in the art, an expression vector comprising anengineered chimeric nucleic acid which encodes both ASIC2A and ASIC3polypeptides as a single tandem polypeptide delimited by the initiationmethionine of the first subunit and the stop of the second subunit. Thefollowing example illustrates this method: The full length codingsequence of human ASIC3 is subcloned into pCDN3 between HindIII andEcoRI sites (plasmid A), while ASIC2A coding nucleic acid sequence issubcloned between EcoRI and XbaI (plasmid B). An ASIC3-specific forwardprimer just upstream of a natural NcoI site (SEQ ID NO:9) is paired witha mutagenic oligonucleotide primer designed to eliminate the stop codonand add an artificial EcoRI site (SEQ ID NO:10). A 570 bp fragment isamplified by PCR using the proof-reading DNA polymerase pfu(Stratagene). Following digestion with NcoI and EcoRI, the purifiedfragment is then back-cloned into the ASIC3-containing plasmid A inreplacement of the corresponding wild type fragment. In the second step,the above mutated ASIC3 full length nucleic acid is cut out from thevector with HindIII and EcoRI and subcloned immediately upstream and inframe of the ASIC2A coding sequence of plasmid B. A number of differentstrategies can be designed to prepare similar constructs and theprevious example is intended solely to illustrate this method and not tolimit its scope in any way. The method also encompasses constructs whereASIC2A is placed upstream of ASIC3 as well as constructs where more thantwo subunits are attached together in any pertinent combination andsynthesised as a single polypeptide.

Example 2 Expression of Functional ASIC-2S.2 Channels in Xenopus laevisOocytes

[0188] According to method 1 described above, nuclei of Xenopus oocytesare injected with ASIC2A and ASIC3 cDNAs separately subcloned intopcDNA3 expression vector (1-5 ng). Control oocytes are injected withH₂O. Oocytes are maintained at 18° C. in modified Barth's solution.Proton-activated currents are measured by two-electrode voltage clamp1−3 days after injection. During voltage clamp (-60 mV/−100 mV)),oocytes are bathed in 116 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 10 mM aceticacid and 5 mM Hepes (pH 7.4 with NaOH). To determine proton-gating, bathsolution is quickly switched to a solution of pH<7.4 for 10 sec, thenreturned to bath solution for washout. The stimulating solution isprepared by lowering the pH of the original bath solution withhydrochloric acid. The osmolality of the solutions is verified with anosmometer and corrected with mannitol or choline chloride. To documentionic selectivity, NaCl is replaced with LiCl or KCl. Current-voltagerelationships are determined by stepping from a holding potential of −60mV to potentials between −100 and +60 mV for 10 seconds before andduring stimulation with low pH solution. FIGS. 1A (human) and 1B (rat)compare proton-activated currents carried by homomultimeric ASIC2A andASIC3 receptors to the heteromultimeric ASIC-2S.2 receptors (ASIC2a+3).The much greater amplitude and the consistent biphasic profile of thecurrent recorded in co-injected oocytes clearly reveals the existence ofa novel proton-gated channel resulting from the assembly of ASIC2A andASIC3. Species differences are also noteworthy. Indeed, using humansubunits, both the early fast and sustained currents are stronglypotentiated, while in the case of rat, the major effect appears to be onthe sustained current only.

[0189]FIG. 2 compares pH-response curves of homomultimeric andheteromultimeric ASIC channels of the present invention. The oocyteexpression system is also used to test and screen for compounds withagonist or antagonist activity. FIGS. 5A and 5B illustrate thisprincipal by showing the inhibitory effects of amiloride and gadoliniumon proton-activated currents.

Example 3 Tissue Distribution of ASIC2A and ASIC3 Transcripts usingRT-PCR

[0190] To document the co-expression of ASIC2A and ASIC3 mRNA in varioustissues, specific oligonucleotide primers are designed and used in aduplex RT-PCR protocol. Fragments of 470 and 340 bp are amplified,respectively, with ASIC2A-specific (SEQ ID NO: 11 and SEQ ID NO: 12) andASIC3-specific (SEQ ID NO:13 and SEQ ID NO:14) primers, enabling theco-amplification of both fragments from a single sample. The reaction iscarried out with the EXPAND long-template polymerase mix, containingboth Taq and Pwo polymerases (Roche), according to the manufacturer'sinstructions. Briefly, the reaction mix includes: dNTPs 0.5 mM, forwardand reverse primers 1 μM each, RT-cDNA template 5 μL, 10× PCR buffer 5μL and polymerase enzyme mix 0.75 μL, all in a final volume of 50 μL.Samples are kept at 4° C. and the enzyme mix is added last. Tubes arethen immediately transferred to the thermocycler preheated to 94° C.,after which cycling is launched. Typical cycling conditions are asfollows: Initial denaturation step: 2 min at 94° C., than 40 cycles of45 sec at 94° C., 45 sec at 58° C. and 2 min at 72° C., followed by afinal extension step of 10 min at 72° C. RT-cDNAs are either commercial(Clontech) or prepared from RNA or mRNA either with Superscript orThermoscript enzyme mixes, according to the manufacturer's directions(Gibco Life Sciences). RNA and mRNA are prepared using standardmolecular biology protocols, such as decribed in Maniatis et al., (seeabove) or using commercially available kits, such as the S.N.A.P. totalRNA isolation kit, Fast Track 2.0 and micro Fast Track 2.0 mRNAisolation kits (InVitrogen). An example of tissue distribution ofhASIC2A and hASIC3 mRNA expression appears in FIG. 4. Trigeminal gangliaare among tissues with the highest coexpression, suggesting that theheteromultimeric ASIC-2S.2 channel might be involved in pain and/orsensory transmission. This constrasts with previous results reported forrat where ASIC2A is apparently not expressed in sensory neurons.

Example 4 Co-Localisation of ASIC2A and ASIC3 Transcripts Demonstratedby in Situ Hybridization

[0191] Hybridization probes derived from SEQ ID NO:1, SEQ ID NO:3, SEQID NO:5 and/or SEQ ID NO:7 are employed to screen cDNAs, genomic DNAs,or mRNAs. An example of such use is in situ hybridization to mRNAs.Briefly, a 278 bp fragment corresponding to nucleotides 181-459 of ratASIC2A (Ser⁶¹-Met¹⁵³) is subcloned between Sac I and Sph I sites of thepGEM5zf vector. A 378 bp fragment of rat ASIC3, corresponding tonucleotides 1142-1520 of ASIC3 (Leu³⁸¹-Pro⁵⁰⁷), is subcloned between SacI and Apa I sites of the pGEM5zf vector. Sense and antisense cRNAs aresynthesized with the SP6 and T7 RNA polymerases in the presence of[³²P]-UTP for Northern blot or a mix of [³⁵S]-CTP and [³⁵S]-UTP for insitu hybridization. For the latter, 6 ηm-thick tissue sections are fixedfor 1 hr with 4% formaldehyde in 0.1 M phosphate buffer (pH 7.2) andwashed extensively with phosphate buffered saline (PBS), then reactedwith acetic anhydride in triethanolamine 0.1 M solution. Then, thesections are hybridized overnight at 55° C. using the double [³⁵S]-CTPand [³⁵S]-UTP-labelled cRNA probes. After extensive washing, thesections are dried and exposed to X-ray film for 2-3 days (Marcinkiewiczet al. Neuroscience 1997; 76: 425). FIG. 5 shows an example in ratcerebellum where ASIC2A and ASIC3 positive grains are clearly present onthe same cell type, namely the Golgi cells (GC) of the granular celllayer.

Example 5 Co-Purification of ASIC2A and ASIC3 Subunits

[0192] The existence of a novel heteromultimeric proton-gated ionchannel, initially revealed by electrophysiological data, is furthercorroborated by biochemical data providing direct evidence of theassociation between ASIC2A and ASIC3 subunits through protein/proteininteractions. For this purpose, N- or C-terminal epitope-tagged fusionproteins are constructed with ASIC2A and ASIC3. For the C-terminus,mutagenic oligonucleotide primers eliminate the stop codons of ASIC2Aand ASIC3 and respectively add an artificial XhoI and SaII restrictionsite to the 3′ end. Then, the PCR-amplified full-length ASIC2A and ASIC3cDNAs are subcloned between the artificial EcoRI-XhoI sites into apcDNA3 vector containing an in frame cassette with a FLAG or His₆epitope followed by an artificial stop codon. N-terminal His₆ tagging ofASIC3 is achieved by directly subcloning a full-length EcoRI-NotI ASIC3fragment into the pcDNA3.1/HisA vector (Invitrogen). Similarly, aPCR-amplified full-length ASIC2A cDNA flanked with EcoRI-XhoI sites issubcloned into the pcDNA3.1/HisB vector (InVitrogen) as well as in thepEGFP-C1 vector (Clontech), providing, respectively, a N-terminal His₆-and GFP-tagged ASC2A. All the above ASIC2A and ASIC3 derivatives weredone with human subunits, but a similar approach can also be done forthe rat subunits, or any other species, by anyone skilled in the art.All new tagged receptors are tested for function before performingco-purification experiments. The results from the functional tests aresummarised in FIG. 6B. For co-purification, the following combinationsare either co-injected into oocytes or co-transfected into HEK 293 cellsfor transient expression:

[0193] 1) ASIC2A-N_His₆+ASIC3-C_FLAG

[0194] 2) 2) ASIC2A-N_GFP+ASIC2A-N_His₆

[0195] 3) ASIC2A-N_GFP+ASIC3-N_His₆.

[0196] (Where “N_” indicates N-terminal tagging and “C_” indicatesC-terminal tagging).

[0197] After the required time for protein expression, oocytes or cellsare collected, lysed and membranes solubilized in a TritonX-100-containing buffer (Tinker et al., Cell 1996; 87: 857—Lê et al., JNeuroscience 1998; 18: 7152). Unsolubilized material is removed bycentrifugation and supernatants are incubated with a Ni-NTA-resin(Quiagen) (2 and 3) or an immunoaffinity resin coupled with antiFLAG M2monoclonal antibodies. After several washes, bound proteins are eithereluted with 500 mM imidazole (Ni-NTA) or directly with the SDS-PAGEloading buffer. Proteins separated by gel electrophoresis aretransferred onto nitrocellulose membranes (Amersham) and immunoprobedwith commercially available antibodies against GFP (Green FluorescentProtein) or His₆ epitopes, followed by peroxidase-labelled secondaryantibodies for chemiluminescence detection (ECL kit, Amersham). Resultsfrom these experiments appear in FIGS. 7A (HEK 293 cells) and 7 B(oocytes) and provide for the first time ever the proof of directprotein-protein interactions between ASIC2A and ASIC3 polypeptides.Indeed, both subunits are always purified together, independently ofwhich subunit is initially targeted by the purification step. We havetherefore provided direct biochemical evidence for the existence of thenovel heteromultimeric ASIC-2S.2 receptor.

Example 6 Screening for Compounds Capable of Modulating Ion ChannelActivity and/or Properties

[0198] As described in example 2, voltage clamped oocytes expressing theASIC-2S.2 channels are used to screen for molecules capable ofmodulating, activating or inhibiting channel activity. See FIGS. 3A and3B.

[0199] Alternatively, permanently or transiently transfected or infectedcell lines (e.g. COS, HEK 293) expressing ASIC-2S.2, cultured inmultiwell plates, are loaded with potential- or cation-sensitive(sodium, calcium) dyes and the fluorescence emission is measuredfollowing application of control and low pH buffers (e.g. pH 7.4 and pH5.0). The responses to both buffers in the presence and absence ofcandidate compounds are compared to identify compounds, which stimulate,inhibit or modulate ASIC-2S.2.

[0200] Particular subclasses of channel antagonists are substancescapable of disrupting protein-protein interactions between the subunits,which compose the ASIC-2S.2 channels. These compounds will eitherprevent the association of ASIC2A and ASIC3 into functional channels ordissociate already assembled ASIC-2S.2 receptors by binding to thespecific protein domains responsible for ASIC subunit interactions. Wehave evidence indicating that the intracellular domains of ASIC2A and/orASIC3 are involved in the assembly of functional channels. The C-terminiappear to be particularly important since modifications to this domaingenerates non-functional subunits in both homo- and heteromultimericform. Indeed, as seen in FIG. 6B, all N-terminally tagged ASIC2A orASIC3 retain full function and ability to interact with a functionalcounterpart. In contrast, C-terminal tagging in most cases creatednon-functional, non-interacting subunits. Therefore, fusion proteins areconstructed comprising only the intracellular domains of human ASIC2A orhuman ASIC3. Briefly, C-terminal fragments of ASIC3 and ASIC2A areamplified with mutagenic primers (SEQ ID NO:15 and NO:16 for hASIC3; SEQID NO:17 and NO:18, for hASIC2A) that add an in frame artificialinitiation site at the 5′ end, as well as artificial HindIII and BamHIrestriction sites, used for subcloning of said fragments into pcDNA3expression vectors. The resulting C-terminal nucleic acid and peptidefragments are indicated respectively in SEQ ID NO: 19 and SEQ ID NO: 20,for human ASIC3 and SEQ ID NO 21 and SEQ ID NO: 22 for human ASIC2A.Additionally, N-terminal fragments of ASIC3 and ASIC2A are constructed,by inserting an artificial stop codon with mutagenic primers (SEQ IDNO:23 and its reverse for hASIC3; SEQ ID NO:24 and its reverse forhASIC2A) using the commercially available mutagenesis kit QuickChange(Stratagene™), according to the manufacturer's directions. All fragmentsare inserted into the pcDNA expression vector. The resulting N-terminalnucleic acid and peptide fragments are indicated respectively in SEQ IDNO: 25 and SEQ ID NO: 26 for human ASIC3 and SEQ ID NO: 27 and SEQ IDNO: 28 for human ASIC2A. The inhibitory effects of these fragments onchannel function and/or assembly is tested by co-expressing thesetruncated constructs together with wild-type ASIC2A and/or ASIC3 both inhomomeric and heteromeric combinations (see FIGS. 9A and 9B). Additionalfusion proteins with shorter fragments of the intracellular domains areused to identify the smallest amino acid sequence involved in theinteraction of ASIC2A and ASIC3. This sequence of amino acids isvalidated by the inhibitory and/or disruptive effect of small peptidescorresponding to the identified amino acid sequence, when said peptidesare introduced into hosts expressing the ASIC-2S.2 heteromultimericchannel. A drug screening method based on the identified peptides isused to identify molecules capable of binding to them. Such compoundswill, in turn, bind to the corresponding amino acid sequence present inthe full-length wild-type subunits and inhibit therefore subunitassembly. A number of approaches can be used for this purpose. Forexample, candidate compounds previously arrayed and attached tomulti-well plates are exposed to the above-described peptides, linked toan additional epitope. After washing steps, wells holding compounds thatbind the specific amino acid sequence are revealed with an ELISA-typebased assay using labelled antibodies against the grafted epitope Dataobtained using different concentrations of the peptides are used tocalculate values for the number, affinity, and association constants ofthe interaction.

[0201] A similar approach as described above is also used with theN-terminal fragments of ASIC2A and/or ASIC3. Briefly, mutagenic primersare designed to introduce an artificial stop codon just upstream of thesequence encoding the first putative transmembrane domain of ASIC2A orASIC3. Using commercially available mutagenesis Kits (e.g. QuickChange,Stratagene), the above mutations are incorporated into the plamidscomprising the ASIC2A or ASIC3 nucleic acids. When these mutatedplasmids are introduced into relevant hosts, only the first portioncorresponding to the N-terminal intracellular domain of the ASIC2Aand/or ASIC3 polypeptides is translated. Coexpression of said fragmentswith full length ASIC2A and ASIC3 is done to document their effect onthe assembly of ASIC-2S.2 channels. Drug screening methods based on thesmallest active N-terminal fragment are performed as described above forthe C-terminal fragments.

Example 7 Antisense Molecules

[0202] Antisense molecules to the sequences encoding the ASIC-2S.2components, or any part thereof, are used to inhibit in vivo or in vitroexpression of naturally occurring ASIC-2S.2. Although use of antisenseoligonucleotides, comprising about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. An oligonucleotide based on the coding sequences of eitherASIC2A or ASIC3 are used to inhibit expression of naturally occurringASIC-2S.2. The complementary oligonucleotide is designed from the mostunique 5′ sequence of the coding region and used either to inhibittranscription by preventing binding to the upstream untranscribedsequence or translation of an ASIC2A- or ASIC3-encoding transcript bypreventing ribosomes from binding. Using an appropriate portion of the5′ sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7,an effective antisense oligonucleotide includes any 15-20 nucleotidesspanning the region which translates into the 5′ coding sequence of thepolypeptides of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO:8.

[0203] All publications mentioned in the above specifications are hereinincorporated by reference. Various modifications and variations of thedescribed method and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology, electrophysiology or related fields are intended tobe within the scope of the following claims.

1 28 1 2748 DNA HUMAN ASIC2A 1 ctcttccaca tcaccttggt gtctccctaaataaaaccag ccctccttat cgcctggaaa 60 aaatcaagag ctagagtttg aatgggttttatataaacac tcacccctgt cagcgtgcgg 120 ctgggctctc aggataaact cacagcatctggcgcgatgc ttgccttgcg ttctctcccc 180 tgaacgtcaa ggtttaagca gagcccgaggactgggagct cttctctgaa attcgatcaa 240 cctgaagcca gttgcggaac tgcacggggtcccgatggac ctcaaggaaa gccccagtga 300 gggcagcctg caaccttcta gcatccagatctttgccaac acctccaccc tccatggcat 360 ccgccacatc ttcgtgtatg ggccgctgaccatccggcgt gtgctgtggg cagtggcctt 420 cgtgggctct ctgggcctgc tgctggtggagagctctgag agggtgtcct actacttctc 480 ctaccagcat gtcactaagg tggacgaagtggtggctcaa agcctggtct tcccagctgt 540 gaccctctgt aacctsaatg gcttccggttctccaggctc accaccaacg acctgtacca 600 tgctggggag ctgctggccc tgctggatgtcaacctgcag atcccggacc cccatctggc 660 tgacccctcc gtgctggagg ccctgcggcagaaggccaac ttcaagcact acaaacccaa 720 gcagttcagc atgctggagt tcctgcaccgtgtgggccat gacctgaagg atatgatgct 780 ctactgcaag ttcaaagggc aggagtgcggccaccaagac ttcaccacag tgtttacaaa 840 atatgggaag tgttacatgt ttaactcaggcgaggatggc aaacctctgc tcaccacggt 900 caaggggggg acaggcaacg ggctggagatcatgctggac attcagcagg atgagtacct 960 gcccatctgg ggagagacag aggaaacgacatttgaagca ggagtgaaag ttcagatcca 1020 cagtcagtct gagccacctt tcatccaagagctgggcttt ggggtggctc cagggttcca 1080 gacctttgtg gccacacagg agcagaggctcacatacctg cccccaccgt ggggtgagtg 1140 ccgatcctca gagatgggcc tcgacttttttcctgtttac agcatcaccg cctgtaggat 1200 tgactgtgag acccgctaca ttgtggaaaactgcaactgc cgcatggttc acatgccagg 1260 ggatgcccct ttttgtaccc ctgagcagcacaaggagtgt gcagagcctg ccctaggtct 1320 gttggcggaa aaggacagca attactgtctctgcaggaca ccctgcaacc taacccgcta 1380 caacaaagag ctctccatgg tgaagatccccagcaagaca tcagccaagt accttgagaa 1440 gaaatttaac aaatcagaaa aatatatctcagagaacatc cttgttctgg atatattttt 1500 tgaagctctc aattatgaga caattgaacagaagaaggcg tatgaagttg ctgccttact 1560 tggtgatatt ggtggtcaga tgggattgttcattggtgct agtatcctta caatactaga 1620 gctctttgat tatatttatg agctgatcaaagagaagcta ttagacctgc ttggcaaaga 1680 ggaggacgaa gggagccacg atgagaatgtgagtacttgt gacacaatgc caaaccactc 1740 tgaaaccatc agtcacactg tgaacgtgcccctgcagacg accctgggga ccttggagga 1800 gattgcctgc tgacacccct cgagtcacccagcactccct ccaaacagac cttgaggccc 1860 aagacccagg acaaggaaca gcaagctcaggtgggatggc cccagtgctg gaaagaagca 1920 agagccccct atgcacacat tgcagactagctgcctagac ctcgctccgg ccacgtccaa 1980 cacgacgcat ccttgggccc cgccgtgcgtccctcttagg agagatgagt cacactctgg 2040 aactgtccaa gaacgaacct gccatcacatctcactgcca gatgtataaa gcacctgcat 2100 gctcagactt cttgtggcgc cacctccacgtctgtcttgt acatgacact cctccacgcg 2160 gtttccagtg tccacactgc tgcccgtgcagtgggaccag attccaggtc caaagtcacc 2220 atgaggccac cctggaatca gaactgcacaatcaagaggg aacccatggg actctctgct 2280 acattcagtt cttgtgtcgt ttgtgaaagttcttaacctg cccaaaaacc cccttttccc 2340 caagctgccc ayggggcttc ggcgccaaaggtgacccgcg ccaacctccc tcccccccca 2400 gtgcctrtga cggcggcaca gcagccagcgggtgggggac gcctgtgttc acccatggtg 2460 cccatgtcgt tcttctctcc ctgtgacacagcttgtacag tctgattctt tttatctggg 2520 gtaggggggc ttttatgttt gtccgatggagatttgtttt gttttgcttc attttatgct 2580 tttttatttt agttttgatg ttctgaggtttgctttggtt tttccatttt ctttggcatt 2640 tatttattcg tgcttcaaat cacagtcatattaaaagctg gtcttgtgga aaaaaaaaaa 2700 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaa 2748 2 512 PRT HUMAN ASIC2A 2 Met Asp Leu Lys GluSer Pro Ser Glu Gly Ser Leu Gln Pro Ser Ser 1 5 10 15 Ile Gln Ile PheAla Asn Thr Ser Thr Leu His Gly Ile Arg His Ile 20 25 30 Phe Val Tyr GlyPro Leu Thr Ile Arg Arg Val Leu Trp Ala Val Ala 35 40 45 Phe Val Gly SerLeu Gly Leu Leu Leu Val Glu Ser Ser Glu Arg Val 50 55 60 Ser Tyr Tyr PheSer Tyr Gln His Val Thr Lys Val Asp Glu Val Val 65 70 75 80 Ala Gln SerLeu Val Phe Pro Ala Val Thr Leu Cys Asn Leu Asn Gly 85 90 95 Phe Arg PheSer Arg Leu Thr Thr Asn Asp Leu Tyr His Ala Gly Glu 100 105 110 Leu LeuAla Leu Leu Asp Val Asn Leu Gln Ile Pro Asp Pro His Leu 115 120 125 AlaAsp Pro Ser Val Leu Glu Ala Leu Arg Gln Lys Ala Asn Phe Lys 130 135 140His Tyr Lys Pro Lys Gln Phe Ser Met Leu Glu Phe Leu His Arg Val 145 150155 160 Gly His Asp Leu Lys Asp Met Met Leu Tyr Cys Lys Phe Lys Gly Gln165 170 175 Glu Cys Gly His Gln Asp Phe Thr Thr Val Phe Thr Lys Tyr GlyLys 180 185 190 Cys Tyr Met Phe Asn Ser Gly Glu Asp Gly Lys Pro Leu LeuThr Thr 195 200 205 Val Lys Gly Gly Thr Gly Asn Gly Leu Glu Ile Met LeuAsp Ile Gln 210 215 220 Gln Asp Glu Tyr Leu Pro Ile Trp Gly Glu Thr GluGlu Thr Thr Phe 225 230 235 240 Glu Ala Gly Val Lys Val Gln Ile His SerGln Ser Glu Pro Pro Phe 245 250 255 Ile Gln Glu Leu Gly Phe Gly Val AlaPro Gly Phe Gln Thr Phe Val 260 265 270 Ala Thr Gln Glu Gln Arg Leu ThrTyr Leu Pro Pro Pro Trp Gly Glu 275 280 285 Cys Arg Ser Ser Glu Met GlyLeu Asp Phe Phe Pro Val Tyr Ser Ile 290 295 300 Thr Ala Cys Arg Ile AspCys Glu Thr Arg Tyr Ile Val Glu Asn Cys 305 310 315 320 Asn Cys Arg MetVal His Met Pro Gly Asp Ala Pro Phe Cys Thr Pro 325 330 335 Glu Gln HisLys Glu Cys Ala Glu Pro Ala Leu Gly Leu Leu Ala Glu 340 345 350 Lys AspSer Asn Tyr Cys Leu Cys Arg Thr Pro Cys Asn Leu Thr Arg 355 360 365 TyrAsn Lys Glu Leu Ser Met Val Lys Ile Pro Ser Lys Thr Ser Ala 370 375 380Lys Tyr Leu Glu Lys Lys Phe Asn Lys Ser Glu Lys Tyr Ile Ser Glu 385 390395 400 Asn Ile Leu Val Leu Asp Ile Phe Phe Glu Ala Leu Asn Tyr Glu Thr405 410 415 Ile Glu Gln Lys Lys Ala Tyr Glu Val Ala Ala Leu Leu Gly AspIle 420 425 430 Gly Gly Gln Met Gly Leu Phe Ile Gly Ala Ser Ile Leu ThrIle Leu 435 440 445 Glu Leu Phe Asp Tyr Ile Tyr Glu Leu Ile Lys Glu LysLeu Leu Asp 450 455 460 Leu Leu Gly Lys Glu Glu Asp Glu Gly Ser His AspGlu Asn Val Ser 465 470 475 480 Thr Cys Asp Thr Met Pro Asn His Ser GluThr Ile Ser His Thr Val 485 490 495 Asn Val Pro Leu Gln Thr Thr Leu GlyThr Leu Glu Glu Ile Ala Cys 500 505 510 3 1732 DNA HUMAN ASIC3 3tcgcacgacg cggttctggc catgaagccc acctcaggcc cagaggaggc ccggcggcag 60ccctcggaca tccgcgtgtt cgccagcaac tgctcgatgc acgggctggg ccacgtcttc 120gggccaggca gcctgagcct gcgccggggg atgtgggcag cggccgtggt cctgtcagtg 180gccaccttcc tctaccaggt ggctgagagg gtgcgctact acagggagtt ccaccaccag 240actgccctgg atgagcgaga aagccaccgg ctcgtcttcc cggctgtcac cctgtgcaac 300atcaacccac tgcgccgctc gcgcctaacg cccaacgacc tgcactgggc tgggtctgcg 360ctgctgggcc tggatcccgc agagcacgcc gccttcctgc gcgccctggg ccggccccct 420gcaccgcccg gcttcatgcc cagtcccacc tttgacatgg cgcaactcta tgcccgtgct 480gggcactccc tggatgacat gctgctggac tgtcgcttcc gtggccaacc ttgtgggcct 540gagaacttca ccacgatctt cacccggatg ggaaagtgct acacatttaa ctctggcgct 600gatggggcag agctgctcac cactactagg ggtggcatgg gcaatgggct ggacatcatg 660ctggacgtgc agcaggagga atatctacct gtgtggaggg acaatgagga gaccccgttt 720gaggtgggga tccgagtgca gatccacagc caggaggagc cgcccatcat cgatcagctg 780ggcttggggg tgtccccggg ctaccagacc tttgtttctt gccagcagca gcagctgagc 840ttcctgccac cgccctgggg cgattgcagt tcagcatctc tgaaccccaa ctatgagcca 900gagccctctg atcccctagg ctcccccagc cccagcccca gccctcccta tacccttatg 960gggtgtcgcc tggcctgcga aacccgctac gtggctcgga agtgcggctg ccgaatggtg 1020tacatgccag gcgacgtgcc agtgtgcagc ccccagcagt acaagaactg tgcccacccg 1080gccatagatg ccatccttcg caaggactcg tgcgcctgcc ccaacccgtg cgccagcacg 1140cgctacgcca aggagctctc catggtgcgg atcccgagcc gcgccgccgc gcgcttcctg 1200gcccggaagc tcaaccgcag cgaggcctac atcgcggaga acgtgctggc cctggacatc 1260ttctttgagg ccctcaacta tgagaccgtg gagcagaaga aggcctatga gatgtcagag 1320ctgcttggtg acattggggg ccagatgggc cttttcatcg gggccagcct gctcaccatc 1380ctcgagatcc tagactacct ctgtgaggtg ttccgagaca aggtcctggg atatttctgg 1440aaccgacagc actcccaaag gcactccagc accaatctgc ttcaggaagg gctgggcagc 1500catcgaaccc aagttcccca cctcagcctg ggccccagac ctcccacccc tccctgtgcc 1560gtcaccaaga ctctctccgc ctcccaccgc acctgctacc ttgtcacaca gctctagacc 1620tgctgtctgt gtcctcggag ccccgccctg acatcctgga catgcctagc ctgcacgtag 1680cttttccgtc ttcaccccaa ataaagtcct aatgcatcaa aaaaaaaaaa aa 1732 4 531 PRTHUMAN ASIC3 4 Met Lys Pro Thr Ser Gly Pro Glu Glu Ala Arg Arg Gln ProSer Asp 1 5 10 15 Ile Arg Val Phe Ala Ser Asn Cys Ser Met His Gly LeuGly His Val 20 25 30 Phe Gly Pro Gly Ser Leu Ser Leu Arg Arg Gly Met TrpAla Ala Ala 35 40 45 Val Val Leu Ser Val Ala Thr Phe Leu Tyr Gln Val AlaGlu Arg Val 50 55 60 Arg Tyr Tyr Arg Glu Phe His His Gln Thr Ala Leu AspGlu Arg Glu 65 70 75 80 Ser His Arg Leu Val Phe Pro Ala Val Thr Leu CysAsn Ile Asn Pro 85 90 95 Leu Arg Arg Ser Arg Leu Thr Pro Asn Asp Leu HisTrp Ala Gly Ser 100 105 110 Ala Leu Leu Gly Leu Asp Pro Ala Glu His AlaAla Phe Leu Arg Ala 115 120 125 Leu Gly Arg Pro Pro Ala Pro Pro Gly PheMet Pro Ser Pro Thr Phe 130 135 140 Asp Met Ala Gln Leu Tyr Ala Arg AlaGly His Ser Leu Asp Asp Met 145 150 155 160 Leu Leu Asp Cys Arg Phe ArgGly Gln Pro Cys Gly Pro Glu Asn Phe 165 170 175 Thr Thr Ile Phe Thr ArgMet Gly Lys Cys Tyr Thr Phe Asn Ser Gly 180 185 190 Ala Asp Gly Ala GluLeu Leu Thr Thr Thr Arg Gly Gly Met Gly Asn 195 200 205 Gly Leu Asp IleMet Leu Asp Val Gln Gln Glu Glu Tyr Leu Pro Val 210 215 220 Trp Arg AspAsn Glu Glu Thr Pro Phe Glu Val Gly Ile Arg Val Gln 225 230 235 240 IleHis Ser Gln Glu Glu Pro Pro Ile Ile Asp Gln Leu Gly Leu Gly 245 250 255Val Ser Pro Gly Tyr Gln Thr Phe Val Ser Cys Gln Gln Gln Gln Leu 260 265270 Ser Phe Leu Pro Pro Pro Trp Gly Asp Cys Ser Ser Ala Ser Leu Asn 275280 285 Pro Asn Tyr Glu Pro Glu Pro Ser Asp Pro Leu Gly Ser Pro Ser Pro290 295 300 Ser Pro Ser Pro Pro Tyr Thr Leu Met Gly Cys Arg Leu Ala CysGlu 305 310 315 320 Thr Arg Tyr Val Ala Arg Lys Cys Gly Cys Arg Met ValTyr Met Pro 325 330 335 Gly Asp Val Pro Val Cys Ser Pro Gln Gln Tyr LysAsn Cys Ala His 340 345 350 Pro Ala Ile Asp Ala Ile Leu Arg Lys Asp SerCys Ala Cys Pro Asn 355 360 365 Pro Cys Ala Ser Thr Arg Tyr Ala Lys GluLeu Ser Met Val Arg Ile 370 375 380 Pro Ser Arg Ala Ala Ala Arg Phe LeuAla Arg Lys Leu Asn Arg Ser 385 390 395 400 Glu Ala Tyr Ile Ala Glu AsnVal Leu Ala Leu Asp Ile Phe Phe Glu 405 410 415 Ala Leu Asn Tyr Glu ThrVal Glu Gln Lys Lys Ala Tyr Glu Met Ser 420 425 430 Glu Leu Leu Gly AspIle Gly Gly Gln Met Gly Leu Phe Ile Gly Ala 435 440 445 Ser Leu Leu ThrIle Leu Glu Ile Leu Asp Tyr Leu Cys Glu Val Phe 450 455 460 Arg Asp LysVal Leu Gly Tyr Phe Trp Asn Arg Gln His Ser Gln Arg 465 470 475 480 HisSer Ser Thr Asn Leu Leu Gln Glu Gly Leu Gly Ser His Arg Thr 485 490 495Gln Val Pro His Leu Ser Leu Gly Pro Arg Pro Pro Thr Pro Pro Cys 500 505510 Ala Val Thr Lys Thr Leu Ser Ala Ser His Arg Thr Cys Tyr Leu Val 515520 525 Thr Gln Leu 530 5 2565 DNA RAT ASIC2A 5 caggctctca ggataactcccagtgtctgg cctgatgctt gcctggcgat ctctgccttg 60 aatgccaagg tttaagcagaattcagagga ctgagaactc ttccctgaga tttgatcaac 120 ctgaagccag ttgcagaactgcacagggtc ccgatggacc tcaaggagag ccccagtgag 180 ggcagcctgc aaccttccagtatccagatc ttcgccaata cctccactct ccatggcatc 240 cgccacatct tcgtgtatgggccgctgacc atccggcgtg tgctttgggc agtggccttc 300 gtcggatccc tgggcctgctgctggtggag agctcggaga gggtgtccta ctatttctct 360 tatcagcatg ttaccaaggtggatgaagtg gtggcccaga gcctggtctt cccagctgtg 420 accctctgca acctcaatggcttccggttc tccaggctta ccaccaacga cttgtaccac 480 gctggggagt tgctggccctgctggatgtc aacctacaga ttcccgaccc gcatctggca 540 gaccccacgg tgctggaggccctccgacag aaggccaact tcaaacacta caaaccgaag 600 cagttcagca tgctggagttcctgcaccgg gtaggccatg acctgaagga tatgatgctc 660 tactgcaagt tcaaggggcaggagtgtggg catcaagact tcaccacagt gtttacaaaa 720 tacgggaagt gttacatgtttaactcaggc gaggatggca agccgctgct caccacggtc 780 aaggggggga cgggcaacgggctggagatc atgctggaca ttcagcaaga tgagtacctg 840 cccatctggg gagagacagaggaaacaacg tttgaagcag gagtgaaggt tcagatccac 900 agtcagtctg agccgcctttcatccaagag ctgggctttg gggtggctcc ggggttccag 960 accttcgtgg ccacacaagagcagaggctc acatatctgc ccccaccatg gggggagtgc 1020 cggtcctcag agatgggactcgacttcttt cctgtttaca gcatcacagc ctgtcggatt 1080 gactgtgaga cccgctacatcgtggagaac tgtaactgcc gcatggtcca catgccaggg 1140 gacgcccctt tctgcacccctgagcagcac aaggagtgtg cagagcctgc cctcggtcta 1200 ctggcagaaa aggacagcaattactgtctc tgcaggacac cctgcaacct gacacgctac 1260 aacaaagagc tctccatggtgaagatcccc agcaagacgt cagccaagta cttagagaag 1320 aaatttaaca aatcggaaaaatatatctca gagaacattc ttgttctgga catatttttt 1380 gaggcgctca attacgaaacaattgaacag aagaaggcgt atgaagttgc tgccttactt 1440 ggtgacatcg gtggtcagatgggactgttc attggtgcta gtctcctcac aatactagag 1500 ctctttgatt atatttatgagctgatcaaa gagaagctat tagacctgct tggcaaagaa 1560 gaagaggaag ggagccacgatgagaacatg agcacctgtg acacaatgcc aaaccactct 1620 gaaaccatca gccacactgtgaacgtgccc ctgcagacag ctttgggcac cctggaggag 1680 attgcctgct gacacctctcaggcaacgca gcacctccaa acagacctta aaggcccaag 1740 acctaggaca ggagacagcaagcgcaggtg ggatcgcccc tgacgactga aagaagcaga 1800 gccccccata tgcacacattgcgaacttct gccaaacctc acctggccac atctgacatg 1860 aaccgtcccg ggccctgcgtcatgtccctc gcaggaccga tgagtcgcac tccggaactg 1920 tccaagaact aacctgccatcacatctcac tgccagatgt acaaagcacc tgcatgctca 1980 gacttcttac agcgccacctccacttccga cttgtacgtg atattttctc cgtgcggttt 2040 ccagggccca ctccgctgcccaggcaatgg gaccaggttc cagccccaaa gtcaccctga 2100 gcccagctcc ggaatcgaaactgcacagtc aagaaggaaa ccacagaact ctctacgttt 2160 gatccttgtg ttgtttgtgaccgttcttag ccttgtcctc caaactggcc caaggggcta 2220 ctgcactaaa ggtgaccagtaccaacctcc ttcttttccc agcacccgtg aaggaggtac 2280 agtggcccgg gtgaccccagtatttgtcca tggagccaca ttgttctctc cctgtgacac 2340 agctgtagag tctgattttgttttgttttg ttttgtttag ggcggggact gtttttgttt 2400 gtctatggaa gatttgttttgttccgcttt gtcttacggt cttcggtttt gatgttctaa 2460 ggttcgaatt gggttttccatttttttttt tgagtttatt tattcgtgct tcgaaccaca 2520 gtcatattaa aagctggtcttgtggaaaaa aaaaaaaaaa aaaaa 2565 6 512 PRT RAT ASIC2A 6 Met Asp Leu LysGlu Ser Pro Ser Glu Gly Ser Leu Gln Pro Ser Ser 1 5 10 15 Ile Gln IlePhe Ala Asn Thr Ser Thr Leu His Gly Ile Arg His Ile 20 25 30 Phe Val TyrGly Pro Leu Thr Ile Arg Arg Val Leu Trp Ala Val Ala 35 40 45 Phe Val GlySer Leu Gly Leu Leu Leu Val Glu Ser Ser Glu Arg Val 50 55 60 Ser Tyr TyrPhe Ser Tyr Gln His Val Thr Lys Val Asp Glu Val Val 65 70 75 80 Ala GlnSer Leu Val Phe Pro Ala Val Thr Leu Cys Asn Leu Asn Gly 85 90 95 Phe ArgPhe Ser Arg Leu Thr Thr Asn Asp Leu Tyr His Ala Gly Glu 100 105 110 LeuLeu Ala Leu Leu Asp Val Asn Leu Gln Ile Pro Asp Pro His Leu 115 120 125Ala Asp Pro Thr Val Leu Glu Ala Leu Arg Gln Lys Ala Asn Phe Lys 130 135140 His Tyr Lys Pro Lys Gln Phe Ser Met Leu Glu Phe Leu His Arg Val 145150 155 160 Gly His Asp Leu Lys Asp Met Met Leu Tyr Cys Lys Phe Lys GlyGln 165 170 175 Glu Cys Gly His Gln Asp Phe Thr Thr Val Phe Thr Lys TyrGly Lys 180 185 190 Cys Tyr Met Phe Asn Ser Gly Glu Asp Gly Lys Pro LeuLeu Thr Thr 195 200 205 Val Lys Gly Gly Thr Gly Asn Gly Leu Glu Ile MetLeu Asp Ile Gln 210 215 220 Gln Asp Glu Tyr Leu Pro Ile Trp Gly Glu ThrGlu Glu Thr Thr Phe 225 230 235 240 Glu Ala Gly Val Lys Val Gln Ile HisSer Gln Ser Glu Pro Pro Phe 245 250 255 Ile Gln Glu Leu Gly Phe Gly ValAla Pro Gly Phe Gln Thr Phe Val 260 265 270 Ala Thr Gln Glu Gln Arg LeuThr Tyr Leu Pro Pro Pro Trp Gly Glu 275 280 285 Cys Arg Ser Ser Glu MetGly Leu Asp Phe Phe Pro Val Tyr Ser Ile 290 295 300 Thr Ala Cys Arg IleAsp Cys Glu Thr Arg Tyr Ile Val Glu Asn Cys 305 310 315 320 Asn Cys ArgMet Val His Met Pro Gly Asp Ala Pro Phe Cys Thr Pro 325 330 335 Glu GlnHis Lys Glu Cys Ala Glu Pro Ala Leu Gly Leu Leu Ala Glu 340 345 350 LysAsp Ser Asn Tyr Cys Leu Cys Arg Thr Pro Cys Asn Leu Thr Arg 355 360 365Tyr Asn Lys Glu Leu Ser Met Val Lys Ile Pro Ser Lys Thr Ser Ala 370 375380 Lys Tyr Leu Glu Lys Lys Phe Asn Lys Ser Glu Lys Tyr Ile Ser Glu 385390 395 400 Asn Ile Leu Val Leu Asp Ile Phe Phe Glu Ala Leu Asn Tyr GluThr 405 410 415 Ile Glu Gln Lys Lys Ala Tyr Glu Val Ala Ala Leu Leu GlyAsp Ile 420 425 430 Gly Gly Gln Met Gly Leu Phe Ile Gly Ala Ser Leu LeuThr Ile Leu 435 440 445 Glu Leu Phe Asp Tyr Ile Tyr Glu Leu Ile Lys GluLys Leu Leu Asp 450 455 460 Leu Leu Gly Lys Glu Glu Glu Glu Gly Ser HisAsp Glu Asn Met Ser 465 470 475 480 Thr Cys Asp Thr Met Pro Asn His SerGlu Thr Ile Ser His Thr Val 485 490 495 Asn Val Pro Leu Gln Thr Ala LeuGly Thr Leu Glu Glu Ile Ala Cys 500 505 510 7 1602 DNA RAT ASIC3 7atgaaacctc gctccggact ggaggaggcc cagcggcgac aggcctcaga catccgggtg 60tttgccagca gctgcacaat gcatggtctg ggccacatct ttggccctgg aggcctgacc 120ctgcgccgag ggctgtgggc cacagctgtg ctcctgtcgc tggcggcctt cctctaccag 180gtggctgagc gggttcgcta ctatggggag ttccaccata agaccaccct ggatgagcgt 240gagagccacc agctcacctt cccagctgtg actctgtgta atatcaaccc actgcgccgc 300tcacgcctca cacccaatga cttgcactgg gctggaacag cgctgctggg cctggaccct 360gctgaacatg ctgcctacct tcgtgcactg ggccagcccc ccgcaccacc tggcttcatg 420cccagtccga cctttgacat ggcacaactc tacgccagag ccggccactc ccttgaggac 480atgttgttgg attgccgata ccgtggccag ccctgtgggc ctgagaactt cacagtgatc 540tttactcgaa tggggcaatg ctacaccttc aactctggtg cccacggtgc agagctgctc 600accactccaa agggtggtgc tggcaacgga ctggagatta tgctagatgt acagcaagag 660gagtatctgc ccatctggaa ggacatggaa gagaccccgt ttgaggtggg gatccgagtg 720cagattcaca gccaggatga gccccctgcc attgaccagc tgggcttcgg ggcagcccca 780ggccatcaga cttttgtgtc ctgtcagcag cagcaactga gtttcctgcc accaccctgg 840ggtgactgca ataccgcatc tttggatccc gacgactttg atccagagcc ctctgatccc 900ttgggttccc ccagacccag acccagccct ccttatagtt taataggttg tcgcctggcc 960tgtgagtctc gctatgtggc tcggaagtgt ggctgtcgaa tgatgcatat gcctggaaac 1020tccccagtgt gcagccccca gcagtacaag gactgcgcca gcccagctct ggacgctatg 1080ctgcgaaagg acacgtgtgt ctgccccaac ccgtgcgcta ctacacgcta tgccaaggag 1140ctctccatgg tgcggattcc cagccgcgcg tcagctcgct acctggcccg gaaatacaac 1200cgcagcgagt cctacattac ggagaatgta ctggttctgg atatcttctt tgaggccctc 1260aactatgaag cggtggaaca aaaggcggcc tatgaagtgt cggagctgct gggagacatt 1320gggggacaga tgggactgtt tattggagca agcctgctta ccatccttga gatcctcgac 1380tatctctgtg aggttttcca agacagagtc ctggggtatt tctggaacag aaggagcgct 1440caaaagcgct ctggcaacac tctgctccag gaagagttga atggccatcg aacacatgtt 1500ccccacctca gcctagggcc caggcctcct accactccct gtgctgtcac caagacactc 1560tctgcctccc accgtacctg ttacctcgtc acaaggctct ag 1602 8 533 PRT RAT ASIC2A8 Met Lys Pro Arg Ser Gly Leu Glu Glu Ala Gln Arg Arg Gln Ala Ser 1 5 1015 Asp Ile Arg Val Phe Ala Ser Ser Cys Thr Met His Gly Leu Gly His 20 2530 Ile Phe Gly Pro Gly Gly Leu Thr Leu Arg Arg Gly Leu Trp Ala Thr 35 4045 Ala Val Leu Leu Ser Leu Ala Ala Phe Leu Tyr Gln Val Ala Glu Arg 50 5560 Val Arg Tyr Tyr Gly Glu Phe His His Lys Thr Thr Leu Asp Glu Arg 65 7075 80 Glu Ser His Gln Leu Thr Phe Pro Ala Val Thr Leu Cys Asn Ile Asn 8590 95 Pro Leu Arg Arg Ser Arg Leu Thr Pro Asn Asp Leu His Trp Ala Gly100 105 110 Thr Ala Leu Leu Gly Leu Asp Pro Ala Glu His Ala Ala Tyr LeuArg 115 120 125 Ala Leu Gly Gln Pro Pro Ala Pro Pro Gly Phe Met Pro SerPro Thr 130 135 140 Phe Asp Met Ala Gln Leu Tyr Ala Arg Ala Gly His SerLeu Glu Asp 145 150 155 160 Met Leu Leu Asp Cys Arg Tyr Arg Gly Gln ProCys Gly Pro Glu Asn 165 170 175 Phe Thr Val Ile Phe Thr Arg Met Gly GlnCys Tyr Thr Phe Asn Ser 180 185 190 Gly Ala His Gly Ala Glu Leu Leu ThrThr Pro Lys Gly Gly Ala Gly 195 200 205 Asn Gly Leu Glu Ile Met Leu AspVal Gln Gln Glu Glu Tyr Leu Pro 210 215 220 Ile Trp Lys Asp Met Glu GluThr Pro Phe Glu Val Gly Ile Arg Val 225 230 235 240 Gln Ile His Ser GlnAsp Glu Pro Pro Ala Ile Asp Gln Leu Gly Phe 245 250 255 Gly Ala Ala ProGly His Gln Thr Phe Val Ser Cys Gln Gln Gln Gln 260 265 270 Leu Ser PheLeu Pro Pro Pro Trp Gly Asp Cys Asn Thr Ala Ser Leu 275 280 285 Asp ProAsp Asp Phe Asp Pro Glu Pro Ser Asp Pro Leu Gly Ser Pro 290 295 300 ArgPro Arg Pro Ser Pro Pro Tyr Ser Leu Ile Gly Cys Arg Leu Ala 305 310 315320 Cys Glu Ser Arg Tyr Val Ala Arg Lys Cys Gly Cys Arg Met Met His 325330 335 Met Pro Gly Asn Ser Pro Val Cys Ser Pro Gln Gln Tyr Lys Asp Cys340 345 350 Ala Ser Pro Ala Leu Asp Ala Met Leu Arg Lys Asp Thr Cys ValCys 355 360 365 Pro Asn Pro Cys Ala Thr Thr Arg Tyr Ala Lys Glu Leu SerMet Val 370 375 380 Arg Ile Pro Ser Arg Ala Ser Ala Arg Tyr Leu Ala ArgLys Tyr Asn 385 390 395 400 Arg Ser Glu Ser Tyr Ile Thr Glu Asn Val LeuVal Leu Asp Ile Phe 405 410 415 Phe Glu Ala Leu Asn Tyr Glu Ala Val GluGln Lys Ala Ala Tyr Glu 420 425 430 Val Ser Glu Leu Leu Gly Asp Ile GlyGly Gln Met Gly Leu Phe Ile 435 440 445 Gly Ala Ser Leu Leu Thr Ile LeuGlu Ile Leu Asp Tyr Leu Cys Glu 450 455 460 Val Phe Gln Asp Arg Val LeuGly Tyr Phe Trp Asn Arg Arg Ser Ala 465 470 475 480 Gln Lys Arg Ser GlyAsn Thr Leu Leu Gln Glu Glu Leu Asn Gly His 485 490 495 Arg Thr His ValPro His Leu Ser Leu Gly Pro Arg Pro Pro Thr Thr 500 505 510 Pro Cys AlaVal Thr Lys Thr Leu Ser Ala Ser His Arg Thr Cys Tyr 515 520 525 Leu ValThr Arg Leu 530 9 20 DNA HUMAN ASIC3 9 aggtgttccg agacaaggtc 20 10 31DNA HUMAN ASIC3 10 gcgaattccg agagctgtgt gacaaggtag c 31 11 20 DNA HUMANASIC2A 11 tctttgccaa cacctccacc 20 12 20 DNA HUMAN ASIC2A 12 ctcctgccctttgaacttgc 20 13 18 DNA HUMAN ASIC3 13 agtggccacc ttcctcta 18 14 20 DNAHUMAN ASIC3 14 cagtccagca gcatgtcatc 20 15 29 DNA HUMAN ASIC3 15tccaagctta tgggatattt ctggaaccg 29 16 28 DNA HUMAN ASIC3 16 cgggatccaaagctacgtgc aggctagg 28 17 31 DNA HUMAN ASIC2A 17 tccaagctta tgcttggcaaagaggaggac g 31 18 28 DNA HUMAN ASIC2A 18 cgggatccga gcttgctgtt ccttgtcc28 19 192 DNA HUMAN ASIC3 19 atgggatatt tctggaaccg acagcactcc caaaggcactccagcaccaa tctgcttcag 60 gaagggctgg gcagccatcg aacccaagtt ccccacctcagcctgggccc cagacctccc 120 acccctccct gtgccgtcac caagactctc tccgcctcccaccgcacctg ctaccttgtc 180 acacagctct ag 192 20 64 PRT HUMAN ASIC3 20 MetLeu Gly Tyr Phe Trp Asn Arg Gln His Ser Gln Arg His Ser Ser 1 5 10 15Thr Asn Leu Leu Gln Glu Gly Leu Gly Ser His Arg Thr Gln Val Pro 20 25 30His Leu Ser Leu Gly Pro Arg Pro Pro Thr Pro Pro Cys Ala Val Thr 35 40 45Lys Thr Leu Ser Ala Ser His Arg Thr Cys Tyr Leu Val Thr Gln Leu 50 55 6021 147 DNA HUMAN ASIC2A 21 atgcttggca aagaggagga cgaagggagc cacgatgagaatgtgagtac ttgtgacaca 60 atgccaaacc actctgaaac catcagtcac actgtgaacgtgcccctgca gacgaccctg 120 gggaccttgg aggagattgc ctgctga 147 22 48 PRTHUMAN ASIC2A 22 Met Leu Gly Lys Glu Glu Asp Glu Gly Ser His Asp Glu AsnVal Ser 1 5 10 15 Thr Cys Asp Thr Met Pro Asn His Ser Glu Thr Ile SerHis Thr Val 20 25 30 Asn Val Pro Leu Gln Thr Thr Leu Gly Thr Leu Glu GluIle Ala Cys 35 40 45 23 25 DNA HUMAN ASIC3 23 ggatgtgggc ataggccgtggtcct 25 24 24 DNA HUMAN ASIC2A 24 cgtgtgctgt gagcagtggc cttc 24 25 152DNA HUMAN ASIC3 25 atgaagccca cctcaggccc agaggaggcc cggcggcagccctcggacat ccgcgtgttc 60 gccagcaact gctcgatgca cgggctgggc cacgtcttcgggccaggcag cctgagcctg 120 cgccggggga tgtgggcata ggccgtggtc ct 152 26 46PRT HUMAN ASIC3 26 Met Lys Pro Thr Ser Gly Pro Glu Glu Ala Arg Arg GlnPro Ser Asp 1 5 10 15 Ile Arg Val Phe Ala Ser Asn Cys Ser Met His GlyLeu Gly His Val 20 25 30 Phe Gly Pro Gly Ser Leu Ser Leu Arg Arg Gly MetTrp Ala 35 40 45 27 147 DNA HUMAN ASIC2A 27 atggacctca aggagagccccagtgagggc agcctgcaac cttccagtat ccagatcttc 60 gccaatacct ccactctccatggcatccgc cacatcttcg tgtatgggcc gctgaccatc 120 cggcgtgtgc tttgagcagtggccttc 147 28 44 PRT HUMAN ASIC2A 28 Met Asp Leu Lys Glu Ser Pro SerGlu Gly Ser Leu Gln Pro Ser Ser 1 5 10 15 Ile Gln Ile Phe Ala Asn ThrSer Thr Leu His Gly Ile Arg His Ile 20 25 30 Phe Val Tyr Gly Pro Leu ThrIle Arg Arg Val Leu 35 40

What is claimed is: 1) A protein complex forming a heteromultimericamiloride- and gadolinium-sensitive proton-gated cation channel, wherethe individual components of said heteromultimeric channel include theASIC2A protein of SEQ ID NO:2 or SEQ ID NO:6 and the ASIC3 protein ofSEQ ID NO:4 or SEQ ID NO:8, or any variants thereof having at least 80%identity to the above enumerated amino acid sequences, and said channelbeing activated by protons, acids, low pH solutions. 2) The proteincomplex of claim 1, where said heteromultimeric channel is comprised ofthe ASIC2A protein of SEQ ID NO:2 or SEQ ID NO:6 and the ASIC3 proteinof SEQ ID NO:4 or SEQ ID NO:8, or any variants thereof having at least80% identity to the above enumerated amino acid sequences. 3) Theprotein complex of claim 1 where said heteromultimeric channel iscomprised of the ASIC2A protein of SEQ ID NO:2 or SEQ ID NO:6 and theASIC3 protein of SEQ ID NO:4 or SEQ ID NO:8. 4) A nucleic acid, whichencodes as a single continuous amino acid sequence or single protein atleast two or more individual components, or any variant thereof, of theprotein complex of claim 1, claim 2 or claim
 3. 5) The nucleic acid ofclaim 4, which is capable of hybridizing to SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5 and/or SEQ ID NO:7. 6) The nucleic acid of claim 4, whichcomprises the nucleic acid sequence defined in SEQ ID NO:1 or SEQ IDNO:5. 7) The nucleic acid of claim 4, which comprises the nucleic acidsequence defined in SEQ ID NO:3 or SEQ ID NO:7. 8) A recombinantbiscistronic vector comprising nucleic acids encoding at least twoindividual components of the heteromultimeric channel of claim 1, claim2, or claim
 3. 9) A recombinant biscistronic vector comprising thenucleic acids of SEQ ID NO:1 and SEQ ID NO:3, or any variants thereofhaving at least 80% identity. 10) A recombinant biscistronic vectorcomprising the nucleic acids of SEQ ID NO:5 and SEQ ID NO:7, or anyvariants thereof having at least 80% identity. 11) A recombinantbiscistronic vector comprising the nucleic acids of any of claims 4, 5,6, or
 7. 12) A recombinant vector, comprising the nucleic acid of claim4, 5, 6, or 7 13) The recombinant vector of claim 8, 9, 10, 11, or 12,which is an expression vector. 14) A host comprising the recombinantvector of claim 8, 9, 10, 11, 12, or
 13. 15) A host comprising tworecombinant vectors, V1 or V2 and V3 or V4, of any type or nature (e.g.expression vector) and defined as follows: a) V1 is a recombinant vectorcomprising the nucleic acid of SEQ ID NO:1 or a nucleic acid capable ofhybridizing to SEQ ID NO:1 under stringent conditions. b) V2 is arecombinant vector comprising the nucleic acid of SEQ ID NO:5 or anucleic acid capable of hybridizing to SEQ ID NO:5 under stringentconditions. c) V3 is a recombinant vector comprising the nucleic acid ofSEQ ID NO:3 or a nucleic acid capable of hybridizing to SEQ ID NO:3under stringent conditions. d) V4 is a recombinant vector comprising thenucleic acid of SEQ ID NO:7 or a nucleic acid capable of hybridizing toSEQ ID NO:7 under stringent conditions. 16) A host cell comprising therecombinant vector of claim
 13. 17) A host cell comprising tworecombinant vectors as defined in claim 15 and which are expressionvectors. 18) A process for producing a cell, which produces theindividual components of the heteromultimeric channel of claim 1, claim2, or claim 3, including transforming or transfecting a host cell withthe recombinant vector of claim 13 or the recombinant vectors defined inclaim 17, such that the host cell, under appropriate culture conditions,produces the heteromultimeric channel complex of claim 1, claim 2, orclaim
 3. 19) An antibody immunospecific for the protein complex of claim1, claim 2, or claim
 3. 20) An antibody immunospecific for a domain ofone of the components of the protein complex of claim 1, or claim 2, andsaid domain being involved in the interaction between said components.21) An antibody immunospecific for ASIC2A polypeptides of SEQ ID NO:2 orSEQ ID NO:6, which antibody is capable of inhibiting, preventing ordisrupting the assembly of the individual components of the proteincomplex of claim 1, claim 2, or claim
 3. 22) An antibody immunospecificfor ASIC3 polypeptides of SEQ ID NO:4 or SEQ ID NO:8, which antibody iscapable of inhibiting, preventing or disrupting the assembly of theindividual components of the protein complex of claim 1, claim 2, orclaim
 3. 23) A hybridoma producing an antibody as defined in claims 19,20, 21, or
 22. 24) A method for the treatment of a subject in need ofenhanced activity or expression of the heteromultimeric channel complexof claim 1, claim 2 or claim 3, comprising: (a) administering to thesubject a therapeutically effective amount of an agonist to saidheteromultimeric channel; and/or (b) providing to the subject apolynucleotide of claim 4, 5, 6, or 7 in a form so as to effectproduction of said heteromultimeric channel complex activity in vivo.(c) providing to the subject a polynucleotide of SEQ ID NO:1 and/or SEQID NO: 3 in a form so as to effect production of said heteromultimericchannel complex activity in vivo. 25) A method for the treatment of asubject having need to inhibit activity or expression of theheteromultimeric channel complex of claim 1, claim 2 or claim 3,comprising: (a) administering to the subject a therapeutically effectiveamount of an antagonist to said heteromultimeric channel; and/or (b)administering to the subject a nucleic acid molecule that inhibits theexpression of the nucleotide sequence encoding an essential component ofsaid heteromultimeric channel; and/or (c) administering to the subject atherapeutically effective amount of a polypeptide that competes withsaid heteromultimeric channel for its ligand. (d) administering to thesubject a therapeutically effective amount of a substance that inhibits,prevents and/or disrupts the assembly of the individual componentscomprising said heteromultimeric channel. 26) A process for diagnosing adisease or a susceptibility to a disease in a subject related to theexpression or activity of the heteromultimeric channel of claim 1, claim2 or claim 3 in a subject, comprising: (a) determining the presence orabsence of a mutation in the nucleotide sequence encoding a component ofsaid heteromultimeric channel in the genome of said subject; and/or (b)analyzing for the presence or amount of heteromultimeric protein complexexpression in a sample derived from said subject 27) A method forscreening for agonists of the heteromultimeric channel of claim 1, claim2, or claim 3, comprising: (a) putting cells produced by claim 16 orclaim 17 in contact with candidate compound(s); and (b) determiningwhether a candidate compound induces or modulates a biological activitytransduced by said heteromultimeric channel complex; or c) determiningwhether a candidate compound induces inward currents or modulatesproton-induced inward currents transduced by said heteromultimericchannel complex. 28) An agonist identified by the method of claim 27.29) An agonist of claim 28, which is, or is an adjuvant to, anantidepressant, a desensibilising agent, an antipruritic, an analgesic(e.g by neurodegeneration of trigeminal neurons), achemotherapeutic/antineoplastic agent, an antiparkinsonian agent, anigro-strial stimulant, an antipsychotic, a psychotherapeutic agent, arespiratory and cerebral stimulant, a cognitive stimulant, memorystimulant, a promoter of neuronal regeneration, a stimulant of cellgrowth and/or proliferation, an insecticide, a pesticide and/or ananthelmintic, or any combination thereof. 30) The method for screeningfor antagonists of the heteromultimeric channel of claim 1, claim 2, orclaim 3, comprising: (a) putting cells produced by claim 16 or claim 17in contact with a low pH solution (pH<7.4) or any other agonist,including agonist of claim 28, and (b) determing whether the signalgenerated by protons and/or said agonist is modulated, diminished orabolished in the presence of candidate compound(s). 31) An antagonistidentified by the method of claim
 30. 32) An antagonist of claim 31,which is, or is an adjuvant to, an analgesic, an antipyretic, anantipruritic, an anxiolytic, sedative or hypnotic, a psychotherapeuticagent, an anticonvulsant, a neuroprotectant (e.g against central and/orexcitotoxicity, such as in ischemia, stroke . . . ), a general and/orlocal anesthetic, a hypotensive agent, a muscle relaxant, anantidiarrhea agent, an antacid, or any combination thereof.